Zeiss Axioskop Research Articles

Recurrent ligneous conjunctivitis after cataract surgery in a 67-year-old male patient: a case report

BackgroundLigneous conjunctivitis is a rare form of chronic pseudomembranous conjunctivitis which usually starts during infancy. We report on an unsual case of recurrent ligneous conjunctivitis after cataract surgery in a 67-year-old male patient.MethodsThe equipment used for the slit-lamp images was a Haag Streit slit lamp BX900 Sn 00,406 with 16 × magnifications. The used batch number of the camera was sn00406 and the software was from the company CCS Pawlowski Merge Eye. There were no filters used. The images were saved with a resolution of 300 DPI. Neither downstream nor averaging was used to enhance the resolution of the image in the case presentation section or the figure legend.The equipment used for the cross-sectional histologic images was a Zeiss Axioskop 40 microscope with an objective lens Zeiss A-Plan × 20/0.45 (Zoom 6.3 × TV 2/3″″C). The used camera was AxioCam MRc5 and the software was ZEN 3.2. The cross-sectional histologic images were saved with a resolution of 2584 × 1936 Pixels. Neither downstream nor averaging was used to enhance the resolution of the image in the case presentation section or the figure legend.Case presentationThis is a rare case report of ligneous conjunctivitis in a 67-year-old male patient who presented a recurrent conjunctival granuloma after five excisions following cataract surgery in his left eye. We performed a tumor excision with free conjunctival autograft. The histology showed a fibrin crust including macrophages, granulocytes, lymphocytes, and reactively altered squamous cell nests. These findings were consistent with a ″pseudomembrane in conjunctivitis lignosa″. We administered a topical combination of plasminactivator, heparin, cortisone and cyclosporine.ConclusionThis treatment with the combination of plasminactivator, heparin, cortisone and cyclosporine has proven to be effective in preventing the recurrence of ligneous conjunctivitis.

Intratumoural Effector Cell Subpopulations in Breast Cancer: a Literature Review and Own Data Report

Breast cancer (BC) is most prevalent female malignancy worldwide. Despite advances in BC diagnosis and progress in drug therapy, a series of challenges associated with emergent tumour resistance causing the disease escalation still remain. Immune evasion is among the driving forces of tumour resistance against modern treatments, which promotes world-active research into the mechanisms of tumour—immune interaction.Tumour microenvironment is known to contribute greatly to the nature of this interaction. Immune cells are constitutive of tumour microenvironment as tumour-associated macrophages, myeloid-derived suppressor cells and tumour-infi ltrating lymphocytes. Tumour-infi ltrating lymphocytes are represented by B-, T- and NK-cells, which localisation and subpopulation structure in tumour may possess a prognostic and clinical significance. Th e infi ltration density by certain effector cell types prior to chemotherapy is an important predictor of patient survival. Putting otherwise, the presence of effector lymphocyte subpopulations in tumour defi nes the strength of antitumour immunity and may establish the success of drug treatment.This study analysed the infiltration levels of CD3, CD4, CD20 and CD38 lymphocytes in several molecular BC subtypes. Tumour immunophenotyping was performed in cryosectioning and immunofl uorescence assays with a ZEISS AXIOSKOP microscope, Germany. We analysed 96 luminal BC (37 subtype A (38.5 %), 52 B-Her2-negative subtype (54.2 %), 7 B-Her2-positive subtype (7.3 %)) and non-luminal BC samples (3 HER2+ subtype (14.3 %), 18 triple-negative subtype (85.7 %)). The infiltration and antigen expression patterns have been assessed. Analyses of tumour-infi ltrating subpopulations revealed lower infiltration in luminal BC vs. other subtypes, albeit at no significance.

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Dissection‐Based Investigation of Variations in Major Gross Arterial Supply to the Trigeminal Ganglion

Introduction & Objective The trigeminal nerve (CN V) primarily conveys afferent sensory input from the anterolateral aspect of the face and scalp via its three main branches – the ophthalmic nerve (CN V1), maxillary nerve (CN V2), and mandibular nerve (CN V3). The 20,000-35,000 primary afferent pseudounipolar neurons of CN V1-3 converge at the trigeminal ganglion (TG) where the neurons’ cell bodies are positioned. The TG is located in Meckel's cave (cavum trigeminale), an intracranial area along the sphenoid bone deep to the meningeal layer of dura mater just anterior to the petrous apex. Four main arterial sources have been reported to perfuse the TG – the accessory meningeal artery (AMA), a branch of middle meningeal artery (MMA), a branch of internal carotid artery (ICA), and a tentorial branch of the superior cerebellar artery (SCA). The reported rate for each potential arterial supply remains variable. The objective of this study was to identify the arterial source of the TG via human cadaveric dissection and quantify the rate of occurrence. Materials & Methods Right and left trigeminal ganglia were examined on 29 embalmed human cadavers. Origins of gross arterial supply traversing the deep surface of the ganglia were identified and recorded. Arterial supply was photographed (iPad Pro, Apple Inc.) and image color contrast was utilized to aide structure identification (Adobe Photoshop 20.0.9). TG tissue samples including the major vessel(s) were collected, paraffin embedded, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). Major arteries and normal microvasculature were verified with light microscopy (Zeiss Axioskop, Model 45 14 85) and photographed (AmScope Digital Camera, Model MU1003). Results Blood supply to 28 TG was inconclusive due to compromised vessel integrity from a previous course dissection. Of the remaining 30 TG, 56.6% were supplied by a branch of ICA, 16.7% by AMA, and 10.0% by a branch of MMA. No TG were supplied by the tentorial branch of SCA. Anastomotic connections were discovered between MMA/ICA and AMA/ICA for 10.0% and 6.7% of TG supply, respectively. Three of the five AMAs were noted to course through separate foramen in the basicranium instead of the foramen ovale as expected. H&E histological examination confirmed artery structure and verified expected arteriole and capillary microvasculature throughout the TG. Conclusions & Significance In both exclusive and anastomotic form, the majority of TG arterial supply originated from the ICA. Supply from the AMA, MMA, and SCA occurred less often but still merit significance. The results of this study reflect known rates of TG arterial supply. Gross imaging from this study may supply medical educators with new resources to facilitate TG dissection in gross dissection laboratories and may also provide diagnosticians and cardiovascular surgeons with better understanding of TG arterial supply variations that could benefit diagnosis, surgical approaches, and outcomes. In case of blockage, stenosis, or ligation, anastomotic supply would provide anatomical insurance to the TG but warrants further study.

An Overview of Mites on Grapevine and the Discovery of a new Phytoseiidae Species; Typhlodromus (Typhlodromus) spiceae

The common grapevine (Vitis vinifera L.) is the main species used for wine making, with South Africa being one of the top wine exporting countries. Grapevine is vulnerable to a range of pests, includingmites. We present an overview of phytophagous and predatory mites on grapevine in South Africa anddescribe a new phytoseiid species which was discovered in the winelands region of Wellington, SouthAfrica. Grapevine shoots with leaves were collected over two growing seasons at four farms in Wellington.A nursery, mother block and a commercial vineyard were selected on each farm. The mites were removedwith a mite brushing machine and slide mounted. The Phytoseiidae Database, as well the most recentrelevant literature were used in confirming the new species. Illustrations of the new species were madeusing photographs taken by Zeiss Axioskop TM Research that included a Zen Soft Imaging System. Thenew Typhlodromus species, Typhlodromus (Typhlodromus) spiceae sp.n., is set apart from closely relatedspecies by setae Z4 being 0.7 times the length of Z5 and by having a short, saccular and thick-walledspermathecal. This species was found together with one phytophagous mite species (Brevipalpus lewisi)and several predatory mite species (Typhlodromus praeacutus, Typhlodromus saevus, Eusieus addoensis). Akey to identify females of the South African species of Typhlodromus (Typhlodromus) is provided.

Different aspects of studying a diatom Cylindrotheca closterium (Ehrenberg) Reimann et Lewin 1964 in natural and laboratory conditions

The article summarizes original and literary data on different aspects of studying Cylindrotheca closterium (Ehrenberg) Reimann et Lewin 1964 in two biotopes – phytoplankton and microphytobenthos – of the Black Sea, the Sea of Azov, and the Sea of Japan for the period from 1976 to 2016. The aim of the work is to present the results of the study mainly of own data on the morphology, systematics and ecology of C. closterium in different seas and under cultivation in the laboratory. Information on the history of the species origin and its nomenclature changes is given. C. closterium belongs to the phylum Bacillariophyta, class Bacillariophyceae, order Bacillariales Hendey 1937, family Bacillariaceae Ehrenb. 1831, genus Cylindrotheca Rabenhorst 1859 emend. Reim. et Lewin 1964. This benthoplanktonic species occurs in the plankton, in littoral and sublittoral zones of the seas. The species is marine and brackish-water; it is a cosmopolite common in different geographical zones of the World Ocean. The results of studying alga by various methods under natural and experimental conditions in light and transmission electron microscopes of C. Zeiss LIBRA-120 are presented. The quantitative data of C. closterium were determined by direct counting of the cells in the Goryaev’ camera (V = 0.9 mm³) in light microscopes BIOLAM L-212, C. Zeiss Axioskop 40 with the program AxioVision Rel. 4.6 at 10×40, 10×100, and Olympus BX41 (Tokyo, Japan) with lenses UPLanF140× and 100×1/30 oil immersion. Cultivation of C. closterium was carried out in the cumulative mode on the nutrient medium F, volume of 1 L under light intensity of 13.7 klx and temperature of +20…+21 °C. Morphology data of this species from different seas were obtained. The average cell sizes of C. closterium are: 25–260 µm length, 1.5–8 µm width; 12–25 fibulae in 10 µm. The results of cultivation in the laboratory conditions showed that the average cell sizes reached 148.17 µm (length) and 8 µm (width) at the temperature of +19…+20 °C and light intensity of 13 klx; length of cells reached 162.12 µm in the exponential phase of growth and 172.07 µm – in the stationary phase. C. closterium has an important practical significance as a source of fucoxanthin, since this alga is intensively cultivated for production of biologically active substances. Our experimental data showed that during laboratory cultivation the fucoxanthin concentration in a diatom biomass can reach 11 mg·g-1 of dry mass. The new data obtained are relevant and important; they can be used in different fields of science and medicine. The seasonal dynamics of population abundance of C. closterium in different ecotopes (epizoon of invertebrates and their food spectra, epiphyton of bottom vegetation, periphyton of the experimental and anthropogenic substrates of the different seas) is presented for the first time. The maximum abundance of the species population (65.6·10³ cells·cm-2) was registered in the epizoon of the mussel Mytilus galloprovincialis Lam. in March at the water temperature of +7.7 °C at a depth of 2.5 m in the Black Sea. The maximum abundance was registered in the epiphyton of green algae (896·10³ cells·cm-2) and in the periphyton of asbestos plates (728·10³ cells·cm-2) in August at the water temperature of +24.5 °C in the Sea of Japan. The abundance dynamics of C. closterium natural populations in the local habitats changed depending on the season, the depth, and the type of substrate. The similarities and differences in the distribution of C. closterium in the sea microphytobenthos are discussed.

Visualization of Nitric Oxide, Measurement of Nitrosothiols Content, Activity of NOS and NR in Wheat Seedlings.

Nitric oxide (NO), is a redox-active, endogenous signalling molecule involved in the regulation of numerous processes. It plays a crucial role in adaptation and tolerance to various abiotic and biotic stresses. In higher plants, NO is produced either by enzymatic or non-enzymatic reduction of nitrite and an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. There are several methods to measure NO production: mass spectrometry, tissue localization by DAF-FM dye. Electron paramagnetic resonance (EPR) also known as electron spin resonance (ESR) and spectrophotometric assays. The activity of NOS can be measured by L-citrulline based assay and spectroscopic method (NADPH utilization method). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). This experimental method describes visualization of NO using DAF-FM dye by fluorescence microscopy (Zeiss AXIOSKOP 2). The whole procedure is simplified, so it is easy to perform but has a high sensitivity for NO detection. In addition, spectrophotometry based protocols for assay of NOS, Nitrate Reductase (NR) and the content of S-nitrosothiols are also described. These spectrophotometric protocols are easy to perform, less expensive and sufficiently sensitive assays which provide adequate information on NO based regulation of physiological processes depending on the treatments of interest.

OR-014 Chronic exercise potentiates anorectic effects of leptin in hypothalamic Pomc neurons

OR-014 Chronic exercise potentiates anorectic effects of leptin in hypothalamic Pomc neurons

Long‐Period Biorhythm Variation in Enamel Microstructure of Pre‐Columbian South Americans

It has been argued that the periodicity (in days) between dental enamel growth lines known as striae of Retzius reflects an autonomic biorhythm regulating metabolism and life history. Therefore, quantifying variation in Retzius periodicity (RP) has been recently used as a tool to provide life history and metabolic information among humans, primates, and other mammals. For example, body size is a major driving factor in life history evolution, and correlates strongly with RP in anthropoid primates but not lemurs.Varying datasets disagree as to whether this particular relationship among anthropoid species extends within species, particularly within modern humans. Moreover, little is known regarding the impact that geography has had on life history evolution among modern human populations. Altitude is one unexamined factor that may have influenced this evolution, as high‐altitude human populations exhibit multiple adaptations to their relatively hypoxic environment, and low oxygen availability has obvious consequences for overall metabolic output. Therefore, this study uses RP variation to compare a coastal (low altitude) and Andean (high altitude) sample of Pre‐Columbian South Americans. We have two major objectives: 1) determine if RP is correlated with body mass within this previously unsampled group, and more importantly; 2) determine if RP differs between coastal and Andean populations, a possible consequence of life history adaptations to altitude.To answer our questions, we gathered teeth from 10 individuals, 5 each from low and high altitude archaeological sites of Peru, all part of the anthropology collection at the American Museum of Natural History. Teeth were cleaned, embedded in acrylic resin, thin‐sectioned, and polished according to standard dental histology protocols. They were imaged in circularly polarized light using a Zeiss Axioskop II microscope. Photomicrographs of dental enamel were used to count the number of 24‐hour growth lines (cross‐striations) between striae of Retzius, indicating the number of days taken to form each successive stria (RP). An average of cross‐striation breadths was also divided into an average of striae of Retzius breadths in the same region of each tooth to verify the counted value. For each individual, orbital height and foramen magnum length were used as proxy variables to assess body size. Individuals were sexed based on standard cranial characteristics.Regressions of RP against body size proxies demonstrate a statistically significant negative correlation in the total combined sample. There is no significant difference in RP variation between the sexes, nor in body size between low and high altitude populations. However, the higher altitude population has significantly longer RP values. In conclusion, our analysis suggests that as predicted, an intraspecific relationship exists for RP and body size among our sample, and that altitude elicits as yet unidentified life history/metabolic differences expressed in RP, warranting further study. The high vs. low altitude pattern appears to be independent of the relationship we find between body size and RP in studies of other human populations.Support or Funding InformationFGCU Professional Development GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An improved method for detecting telomere size differences in T-lymphocyte interphases from older people with Down syndrome with and without mild cognitive impairment.

Telomere size (quantified by fluorescence intensity and physical lengths) in short-term T-lymphocyte cultures from adults with Down syndrome (DS) with and without mild cognitive impairment (MCI-DS) or dementia was compared. For these studies, dementia status was determined based on longitudinal assessments employing a battery of cognitive and functional assessments developed to distinguish adult-onset impairment from preexisting developmental disability. In the course of our studies using a MetaSystems Image Analyzer in combination with ISIS software and a Zeiss Axioskop 2, we found that Fluorescein isothiocyanate (FITC) telomere fluorescence referenced to chromosome 2-identified FITC probe fluorescence as a nontelomere standard (telomere/cen2 ratio) showed great promise as a biomarker of early decline associated with Alzheimer’s disease (AD) in this high-risk population. We have now obtained a cen (2) CY3 probe that can clearly be distinguished from the blue–green FITC interphase telomere probe, providing a clear distinction between telomere and centromere fluorescence in both interphase and metaphase. We used FITC/CY3 light intensity ratios to compare telomere length in interphases in adults with DS with and without MCI-DS or dementia. Five age-matched female and five age-matched male pairs (n = 10) all showed clear evidence of telomere shortening associated with clinical progression of AD (P < 0.002 – P < 0.000001), with distributions of mean values for cases and controls showing no overlap. We also examined the time needed for microscopy using interphase versus metaphase fluorescence preparations. With interphase preparations, examination time was reduced by an order of magnitude compared with metaphase preparations, indicating that the methods employed herein have considerable practical promise for translation into broad diagnostic practice.

PERK Activation Preserves the Viability and Function of Remyelinating Oligodendrocytes in Immune-Mediated Demyelinating Diseases

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-γ, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

TARP‐associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states

Fast excitatory synaptic transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs), whose biophysical properties are dramatically modulated by the presence of transmembrane AMPAR regulatory proteins (TARPs). To help construct a kinetic model that will realistically describe native AMPAR/TARP function, we have examined the single-channel properties of homomeric GluA1 AMPARs in combination with the TARPs, γ-2, γ-4 and γ-5. In a saturating concentration of agonist, each of these AMPAR/TARP combinations gave rise to single-channel currents with multiple conductance levels that appeared intrinsic to the receptor-channel complex, and showed long-lived subconductance states. The open time and burst length distributions of the receptor complexes displayed multiple dwell-time components. In the case of γ-2- and γ-4-associated receptors, these distributions included a long-lived component lasting tens of milliseconds that was absent from both GluA1 alone and γ-5-associated receptors. The open time distributions for each conductance level required two dwell-time components, indicating that at each conductance level the channel occupies a minimum of two kinetically distinct open states. We have explored how these data place novel constraints on possible kinetic models of TARP-associated AMPARs that may be used to define AMPAR-mediated synaptic transmission.

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Control of breathing and blood pressure by parafacial neurons in conscious rats

The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n = 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABA(A) agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min(-1) kg(-1) with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats.

Characterization of a Novel Activated Ran GTPase Mutant and Its Ability to Induce Cellular Transformation

Ran (Ras-related nuclear) protein, a member of the Ras superfamily of GTPases, is best known for its roles in nucleocytoplasmic transport, mitotic spindle fiber assembly, and nuclear envelope formation. Recently, we have shown that the overexpression of Ran in fibroblasts induces cellular transformation and tumor formation in mice (Ly, T. K., Wang, J., Pereira, R., Rojas, K. S., Peng, X., Feng, Q., Cerione, R. A., and Wilson, K. F. (2010) J. Biol. Chem. 285, 5815-5826). Here, we describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Furthermore, Ran(K152A) expression in the human mammary SKBR3 adenocarcinoma cell line gives rise to an enhanced transformed phenotype and causes a robust stimulation of both ERK and the N-terminal c-Jun kinase (JNK). Microarray analysis reveals that the expression of the gene encoding SMOC-2 (secreted modular calcium-binding protein-2), which has been shown to synergize with different growth factors, is increased by at least 50-fold in cells stably expressing Ran(K152A) compared with cells expressing control vector. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth in NIH-3T3 cells and in SKBR3 cells and also inhibits cell invasion in fibroblasts. Collectively, our findings highlight a novel connection between the hyper-activation of the small GTPase Ran and the matricellular protein SMOC-2 that has important consequences for oncogenic transformation.

Striatal cellular properties conserved from lampreys to mammals

The striatum of the lamprey, the first vertebrate group to appear in evolution, shows striking similarities to that of mammals with respect to histochemical markers, afferent and efferent projections and the effect of dopamine depletion, which leads to hypokinetic motor symptoms. The cellular properties of lamprey striatal neurons were studied here using patch-clamp recordings in acute striatal slices. Sixty-five per cent of recorded neurons were characterised by a prominent inward rectification due to a K+ conductance of the Kir type. They had a ramping response with a long delay to the first action potential due to activation of a low-voltage-activated A-type K+ current. Many such inwardly rectifying neurons (IRNs) had a hyperpolarised resting membrane potential and some had spiny dendrites. The remaining 35% of the neurons (non-IRNs) represent a heterogeneous group, including some with characteristics similar to the fast-spiking interneuron of the mammalian striatum. They showed short-lasting, large after hyperpolarisations (AHPs) and discharged action potentials at high frequency. None of the recorded neurons were spontaneously active but they received GABAergic and glutamatergic synaptic input. The fact that most lamprey striatal neurons display inward rectification indicates that this is a conserved characteristic of striatal neurons throughout vertebrate phylogeny. This is a cellular property of critical importance for the operations of the striatum in mammals.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Conserved Stress-protective Activity between Prion Protein and Shadoo

Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrP(C)), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrPΔHD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrP(C), Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrP(C) and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrPΔN as well as ShoΔN was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although ShoΔHD lost its stress-protective activity, PrPΔHD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrP(C) could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrP(C). Our study reveals a conserved physiological activity between PrP(C) and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrP(C) might act on similar signaling pathways.