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  JoVE Biology

  
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  JoVE Neuroscience

  
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  JoVE Immunology and Infection

  
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  JoVE Developmental Biology

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JoVE Science Education

General Laboratory Techniques

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Basic Methods in Cellular and Molecular Biology

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Essentials of Biology 1

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Essentials of Biology 2

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Essentials of
Neuroscience

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Essentials of Developmental Biology

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Essentials of Behavioral Science

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Essentials of Genetics

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Essentials of Cell Biology

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In JoVE (55)

Other Publications (4)

Articles by Aaron Kolski-Andreaco in JoVE

 JoVE Biology

Mouse Adrenal Chromaffin Cell Isolation

1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, 3Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 4Department of Developmental and Cell Biology, University of California, Irvine (UCI)


JoVE 129

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

 JoVE Biology

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of California, Irvine (UCI), 3Department of Pathology, University of California, Irvine (UCI)


JoVE 134

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

 JoVE Biology

Mouse Dorsal Forebrain Explant Isolation

1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of California, Irvine (UCI), 3Department of Pathology, University of California, Irvine (UCI)


JoVE 135

This video demonstrates the protocol for isolating and culturing explants of the mouse forebrain from embyonic day 12 mice. Procedures for removal of the uterus, embryos from uterus, and dissection of embryos are given. In addition the methodology for transferring these explants onto specialized membranes on which they are cultured is demonstrated. The development of the forebrain can be studied in vitro using this preparations as well as changes in gene expression.

 JoVE Biology

Culture of Mouse Neural Stem Cell Precursors

1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Pathology, University of California, Irvine (UCI), 3Department of Physiology and Biophysics, University of California, Irvine (UCI)


JoVE 152

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

 JoVE Editorial

March 2014: This Month in JoVE - Food-Related Motivation, Pathogens in the Deli, Diagnosing ALS, and Bioengineering Cells into Patterns

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5274

Here's a look at what's coming up in the March 2014 issue of JoVE.

 JoVE Editorial

May 2011: This Month in JoVE


JoVE 3449

The main highlights for our May issue include methods for measuring cognition in zero gravity, isolating mosquito immune cells, engineering recombinant SARS vaccines, and detecting tumors with thermal imaging. In addition, procedures for isolating neural stem cells from human fetal brain and culturing antigen-presenting liver cells will also be released.

 JoVE Editorial

June 2012: This Month in JoVE

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 4467

Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.

 JoVE Editorial

July 2012: This Month in JoVE

1JoVE Content Production, 2Department of Ophthalmology, Massachusetts Eye and Ear


JoVE 5010

Historically, JoVE, The Journal of Visualized Experiments, has focused primarily on biomedical research and has developed subsections for Bioengineering, Clinical and Translational Medicine, Immunology and Infection, and Neuroscience. This July, JoVE launches its Applied Physics section, which includes a range of content from Plasma Physics to Materials Science. We begin the new section with a notable article from Purdue University, where researchers in the Center for Laser-Based Manufacturing are studying.

 JoVE Editorial

August 2012: This Month in JoVE

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5016

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.

 JoVE Editorial

September 2012: This Month in JoVE

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5022

This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.

 JoVE Editorial

November 2012: This Month in JoVE

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5044

In this issue, Oestreicher et al. show us how to isolate magnetotactic bacteria from freshwater samples, and concentrate the bacteria at one end of a glass capillary. The magnetotactic bacteria can then be visualized by light and transmission electron microscopy, and used for various other assays.

 JoVE Editorial

April 2014: This Month in JoVE - Bioengineering for ACL Tears, Neuroscience behind Insect Locomotion, Improved Genetic Engineering of Crops, and Synthesizing Secret Ink

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5323

Here's a look at what's coming up in the April 2014 issue of JoVE.

 JoVE Editorial

January 2015: This Month in JoVE - Introducing JoVE Developmental Biology

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5637

Here's a look at what's coming up in the January 2015 issue of JoVE: The Journal of Visualized Experiments.

JoVE has been revolutionizing scientific publishing since 2006, when we released our first video articles in JoVE Biology. We've grown over the years-adding sections in Neuroscience, Immunology & Infection, Clinical & Translational Medicine, Bioengineering, Applied Physics, Behavior, Chemistry, and Environment.

We are now pleased to introduce a new addition to the JoVE family: JoVE Development, which covers the entire field of developmental biology from the underlying genetic and epigenetic mechanisms to the growth and differentiation of single cells into organs and whole organisms.

In many species, developmental processes offer clues about evolution. For example, the European lancelet (Branchiostoma lanceolatum) has many features of modern fish, but it doesn't have a backbone-so it's emerging as a useful model for studying the divergence of vertebrates from invertebrate ancestors. Hirsinger et al. have developed a method for visualizing embryonic development in the European lancelet. They inject oocytes with mRNAs that encode fluorescent proteins, and following fertilization, developmental processes can be visualized in vivo.

Development also explores interactions between different cell types, and these interactions are fundamental for repairing and regenerating damaged or diseased tissues, like muscle. To study these interactions, Agley et al. take human skeletal muscle biopsies then purify and culture different cell types. They also characterize the cells using immunocytochemical methods that can be adapted to other cell types.

One of the most exciting topics in developmental biology is the engineering of stem cells for experimental and clinical applications. This month we feature two stem cell protocols: Zielins et al. describe the isolation and purification of human adipose-derived stromal cells for bone engineering, and Lei et al. demonstrate how to differentiate embryonic stem cells into embryoid bodies, and then derive cardiac progenitor cells that further differentiate into cardiomyocytes and smooth muscle cells.

Above all, developmental processes are fascinating to visualize, and JoVE Development features techniques for optimizing real-time imaging, such as this protocol for live-imaging of the Drosophila pupal eye. Using image-stabilization techniques, Hellerman et al. compensate for tissue movement and uneven topology to enhance the visualization of the developing Drosophila eye.

You've just had a sneak peek of JoVE's new Development section in the January 2015 issue. Visit the website to see the full-length articles plus our other scientific sections in JoVE: The Journal of Visualized Experiments.

 JoVE Editorial

February 2015: This Month in JoVE - Tracking Down Foodborne Illness, Imaging Baby Brains, and Paying Scientific Attention to Attention

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5675

Here's a look at what's coming up in the February 2015 issue of JoVE: The Journal of Visualized Experiments.

In JoVE Environment we have a method for tracing outbreaks of food poisoning to insects. Everyone knows that insects can deposit disease-causing organisms on our food. Not surprisingly, insects are important vectors of foodborne illnesses caused by pathogens like Salmonella, E. coli, and Listeria. However, it's hard to link an outbreak of food poisoning to a specific type of insect. This is because individual insects are not usually collected aseptically in environmental sampling programs. Therefore, Pava-Ripoll et al. from the U.S. Food and Drug Administration take microbial samples from individual flies under aseptic conditions and use PCR to detect specific pathogens. PCR-positive samples are then confirmed by plating on selective or differential media and through commercial biochemical assays. Using this method, public health officials can better determine how insects contribute to foodborne outbreaks.

JoVE's Clinical & Translational Medicine section features cranial ultrasound-an indispensable technique for safely imaging the neonatal brain. Ecury-Goossen et al. demonstrate optimal settings that provide better imaging quality, and color Doppler techniques for visualizing intracerebral vessels. They also demonstrate how alternate acoustic windows can improve detection of brain injuries. These advances in cranial ultrasound have improved its diagnostic value-allowing timely therapeutic intervention.

In JoVE Behavior we have two articles that deal with attention. One involves attentional set shifting, or the ability to direct attention to informative cues and away from irrelevant ones. Heisler et al. perform this test in mice that have been trained to dig in pots for a food reward. The test cues are different digging materials and different scents. The mice learn to pay attention to a single relevant cue to find their food. The neural circuits behind attentional set shifting are highly conserved between rodents and humans; therefore, this model can be used to preclinically evaluate cognitive deficits and potential therapies.

In another article in JoVE Behavior, Yung et al. perform two well-known attention tests in an online platform. The multiple object tracking (MOT) task studies the motion-based tracking of multiple objects by the visual system, and the Useful Field of View (UFOV) Task assesses attention and processing speed of visual stimuli from a brief glance. Our authors collected data from over 1,700 participants in a Massive Online Open Course. The results were highly consistent with controlled laboratory-based measures of the same tests, showing the usefulness of behavior studies done entirely online.

You've just had a sneak peek of the February 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

 JoVE Editorial

March 2015: This Month in JoVE - Solving Crime with Science, Applying Technology to Understand Trees, and Studying Protein Synthesis on a Chip

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5702

Here's a look at what's coming up in the March 2015 issue of JoVE: The Journal of Visualized Experiments.

In JoVE Environment, we really love trees-especially for the data stored in tree rings, which are not only valued by historians, geologists, and archeologists, but also environmental scientists who study how forest ecosystems respond to environmental changes. Gärtner et al. expand on traditional tree ring research with new protocols for sampling, preparing, and analyzing wooden specimens. They also present a way to resolve digital images for time-series analyses. These techniques will help scientists understand the effects of specific environmental factors on trees and other woody plants.

In JoVE Biology, we have two protocols that can be used in forensic investigations.

When individuals touch objects or other individuals, they can transfer cellular material that contains traces of DNA. But recovering this DNA from a forensic sample is like finding a needle in a haystack. Therefore, Grüner et al. use a targeted approach, zeroing in on potential biological material under a microscope, and amplifying DNA microsatellite markers called short tandem repeats (STRs). Using bioparticles collected from various objects, they successfully obtain useful genetic profiles from "touch DNA" evidence.

Also in JoVE Biology, scientists have been collecting blood on filter paper for diagnostic and forensic analyses for over a century. But after all this time, protocols for preparing and processing dried blood spots remain poorly standardized. So Farash et al. present a comprehensive, step-by-step protocol for collecting blood, preparing blood spots; drying and storing the filter papers, and eluting the blood for analysis. They demonstrate the effectiveness of this protocol by detecting markers of various viral infections on an automated analytical platform, and in a pilot study for detecting viral infections in active drug users.

In JoVE Bioengineering, all the extrinsic "noise" in living cell systems can complicate the study of specific cellular processes. So scientists use cell-free systems to study the dynamics of biological reactions, such as the intrinsic noise of protein synthesis. Norred et al. present a microfabricated device that can confine cell-free protein synthesis to femtoliter-scale reaction volumes. They demonstrate how to use this device to measure the noise of protein expression in a confined cell-free environment.

You've just had a sneak peek of the March 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

 JoVE Editorial

April 2015: This Month in JoVE - Studying Locomotion in Drunken Worms, Preserving Human Liver for Transplantation, and Visualizing Bacterial Swarms

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5707

Here's a look at what's coming up in the April 2015 issue of JoVE: The Journal of Visualized Experiments.

In JoVE Neuroscience, it is clear that alcohol has profound effects on the brain. But why do different individuals respond so differently to alcohol, and why are some more prone to abuse? Some of the answers lie in our genes. In the nematode worm C. elegans, genes are easy to manipulate, and the neurobiology is simple. So it's an excellent model to study the effects of alcohol on behavior. Davies et al. use copper rings to corral worms with different genotypes on agar plates containing alcohol. They take videos of the worms at set time points and measure the locomotion of each genotype using object recognition software With this procedure, C. elegans can help unravel the molecular basis of alcohol sensitivity and tolerance, as well as the risk of abuse.

In JoVE Medicine, alcohol doesn't just affect the brain; it also affects the liver, and for many patients with alcoholic liver disease, liver transplantation is the only hope. But there is a severe shortage of livers available for transplants. Therefore, Bruinsma et al. present an alternative way to preserve donor livers, which are traditionally stored statically on ice. In this new method, the donor liver is kept alive by a perfusion machine and perfused at 21°C-warmer than ice, but lower than normal body temperatures, which allows the liver to be assessed for function and transplantability. This technique may greatly expand the pool of donor livers available to patients in need of transplants.

In JoVE Biology, one of the most commonly studied modes of bacterial motility is called swarming. However, the reproducibility of swarm plate assays can be difficult to achieve, so Morales-Soto et al. present a method that standardizes parameters (such as moisture content of plates) that affect swarming motility. By using bacterial strains expressing different fluorescent proteins, they can observe swarming competition dynamics between different species in a fluorescent time-lapsed video. This method can help elucidate various aspects of bacterial growth, such as how bacteria colonize different types of surfaces.

In JoVE Behavior, Labots et al. demonstrate the modified hole board. This behavioral testing apparatus consists of a protected box and a board containing small cylinders. In one type of experiment, the board can be baited with tasty food rewards. In some cylinders, the treat is locked under a grid, and in differently colored cylinders, the treat is accessible. Small laboratory mammals (such as rats) are conditioned to the food reward and tested for behaviors such as object recognition or food intake inhibition. This test can help measure unconditioned behavior of small laboratory mammals and analyze their cognitive abilities.

You've just had a sneak peek of the April 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

 JoVE Editorial

This Month in JoVE - Assessing Freezing Tolerance in Plants, Patterning 2D Shapes with DNA, Studying Ischemia In Vitro, Studying Social Cognition in Monkeys

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5727

Here's a look at what's coming up in the May 2015 issue of JoVE: The Journal of Visualized Experiments.

In JoVE Environment, it's the middle of spring in the earth's Northern Hemisphere, so seeds are sprouting and buds are blooming. But for actively growing plants, especially those with no freezing tolerance, a cold snap can be deadly. A hard frost can dramatically alter natural ecosystems and devastate agriculture. Even more concerning, erratic weather patterns related to climate change can cause cold-susceptible plants to bud and bloom too early, then fall victim to a subsequent freeze. To protect plants from freezing, we need a better understanding of the freezing process. Wisniewski et al. describe a protocol to visualize this process using high-resolution infrared thermography (HRIT). Using this technology, scientists can determine how ice forms and propagates, and test various compounds that might alter the freezing process or increase freezing tolerance.

In JoVE Chemistry, the principles of self-assembly have been used to engineer a variety of complex structures from biological and non-biological building blocks. This month, Wei et al. present detailed protocols for the self-assembly of single-stranded DNA tiles into complex 2D shapes with the tiles acting as molecular pixels on molecular canvases. These are visualized by atomic force microscopy. The modular nature of this approach allows it to be scalable, and the tiles can self-assemble into tubes and rectangles of varying sizes. This technique is also highly versatile-enabling the construction virtually any 2D shape.

In JoVE Clinical & Translational Medicine, ischemic stroke happens when a blockage in an artery restricts blood flow to the brain, depriving it of oxygen and nutrients. When the blood returns, reperfusion can cause further damage by disrupting the blood-brain barrier and triggering inflammation and oxidative stress. There are many in vivo models of ischemia-reperfusion injury, and while all disease models have drawbacks, in vivo models tend to be technically challenging, with limited applicability for mechanistic studies. To help overcome some of these limitations, Alluri et al. present an in vitro model of ischemia-reperfusion, which can be used to study the molecular mechanisms of blood-brain barrier dysfunction and signs of oxidative stress.

In JoVE Behavior, Fagot et al. study social cognition in monkeys. In this protocol, monkeys living in social groups have free access to multiple test units. When a monkey enters a testing chamber, and reaches through the hand port, a computer recognizes the monkey through its radio frequency ID (RFID) chip and starts the experiment A touch screen displays the stimulus and a food dispenser delivers the reward. Over several years, this procedure has been shown enhance motivation in the subjects and favor complex learning-particularly the social aspects.

You've just had a sneak peek of the May 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

 JoVE Editorial

June 2015: This Month in JoVE - Celebrating JoVE's 100th Issue

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production


JoVE 5730

Here's a look at what's coming up in the June 2015 issue of JoVE: The Journal of Visualized Experiments.

This month marks the release of JoVE's 100th issue, and we now have 10 scientific sections: Biology, Neuroscience, Immunology & Infection, Medicine, Bioengineering, Engineering, Chemistry, Behavior, Environment, and Developmental Biology.

In the field of Developmental Biology, sometimes you'll come across the term "buffy coat"-which might sound like a pet's coat color, or maybe a manicure, or maybe the leather under-armor worn by 17th Century cavalry. But a buffy coat is a product of centrifuging whole blood, and refers to the cloudy layer containing white blood cells, which forms between the red blood cells and the plasma.

Meraviglia et al. collect the buffy coats and turn peripheral blood mononuclear cells (PBMNCs) into induced pluripotent stem cells (iPSCs). Instead of inducing pluripotency as usual with viral vectors, our authors reprogram the cells using non-integrating plasmids. The resulting iPSC colonies show normal morphology, stable karyotypes, and significant expression of pluripotency markers. As far as we know, this is the first report of deriving virus-free iPSCs from frozen buffy coats, which are commonly stored in biobanks, and could become a valuable source of iPSCs for regenerative medicine.

In JoVE Bioengineering, we know that cells can behave differently depending on the stiffness of the extracellular matrix, which is measured in kiloPascals (kPA). Metastatic cancer cells, in particular, show higher traction stress than non-metastatic cells, so scientists examine tumor cell invasion and metastasis using traction force assays. However, traction data are lacking for primary tumor cells. Therefore, Yakut Ali et al. extract primary cells from human tumors and culture them on hydrogel substrates for traction cytometry. This technique may lead to the use of biophysical parameters, such as tumor cell stiffness and traction, in cancer prognostics.

In JoVE Behavior, we feature protocols that deal with cognition and learning in children. In the experimental paradigm by Sargent et al., a mobile hangs above an infant, and a light-emitting diode (LED) is secured to the infant's leg. When it reaches a certain height, the LED activates the mobile. The infants discover that the mobile will spin and play music if they kick their legs up. A motion capture system records these movements so the learning process can be quantified.

Also in JoVE Behavior, children with speech impairments may benefit from a speech-generating device (SGD), which produces an electronic voice using a synthesizer. Some devices have dynamic interfaces, so users can access new words by changing levels. Cognitive factors can affect the ability to navigate through these levels, so Robillard et al. present a detailed protocol for assessing how cognitive factors contribute to navigational abilities. This could, in turn, guide clinicians to select the appropriate speech-generating device for a patient.

You've just had a sneak peek of the June 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Other articles by Aaron Kolski-Andreaco on PubMed

SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

Small conductance Ca2+-activated K+ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BKCa or KV channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca2+-dependent inactivation of CaV channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K+ channels. SK3-1C may therefore suppress calmodulin-gated SKCa/IKCa channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

A Drosophila Protein Specific to Pheromone-sensing Gustatory Hairs Delays Males' Copulation Attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.

Kv1.3 Channels Are a Therapeutic Target for T Cell-mediated Autoimmune Diseases

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

Modulators of Small- and Intermediate-conductance Calcium-activated Potassium Channels and Their Therapeutic Indications

Calcium-activated potassium channels modulate calcium signaling cascades and membrane potential in both excitable and non-excitable cells. In this article we will review the physiological properties, the structure activity relationships of the existing peptide and small molecule modulators and the therapeutic importance of the three small-conductance channels KCa2.1-KCa2.3 (a.k.a. SK1-SK3) and the intermediate-conductance channel KCa3.1 (a.k.a. IKCa1). The apamin-sensitive KCa2 channels contribute to the medium afterhyperpolarization and are crucial regulators of neuronal excitability. Based on behavioral studies with apamin and on observations made in several transgenic mouse models, KCa2 channels have been proposed as targets for the treatment of ataxia, epilepsy, memory disorders and possibly schizophrenia and Parkinson's disease. In contrast, KCa3.1 channels are found in lymphocytes, erythrocytes, fibroblasts, proliferating vascular smooth muscle cells, vascular endothelium and intestinal and airway epithelia and are therefore regarded as targets for various diseases involving these tissues. Since two classes of potent and selective small molecule KCa3.1 blocker, triarylmethanes and cyclohexadienes, have been identified, several of these postulates have already been validated in animal models. The triarylmethane ICA-17043 is currently in phase III clinical trials for sickle cell anemia while another triarylmethane, TRAM-34, has been shown to prevent vascular restenosis in rats and experimental autoimmune encephalomyelitis in mice. Experiments showing that a cyclohexadiene KCa3.1 blocker reduces infarct volume in a rat subdural hematoma model further suggest KCa3.1 as a target for the treatment of traumatic and possibly ischemic brain injury. Taken together KCa2 and KCa3.1 channels constitute attractive new targets for several diseases that currently have no effective therapies.

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