Drilling down further, I am pleased to present Electro-physiology and related data generated by Aruna and collaborators: hN2 Cells-Electro Phys Data Supplement

hN2-Whole Cell Voltage Clamp

Figure. hN2 cells can produce inward currents that generate action potentials. (A) Isolated hN2 with significant neurite growth 1 week  after plating . This cell was subjected to whole cell voltage clamp utilizing a potassium gluconate based intracellular solution. (B) Voltage gated inward and outward currents were elicited from this cell with depolarizing voltage steps. (C) Inward currents from another cell (potassium gluconate intracellular) were abolished by local application of 1 µM tetrodotoxin (red trace) while outward currents remained. Inward current recovered as TTX washed out of the region (green trace). (D) A different cell which exhibited voltage activated inward currents that inactivated in response to a 50 ms prepulse at different membrane potentials. The experiment was done 27 days after the removal of bFGF. A cesium gluconate based intracellular solution was used for this experiment to block outward potassium currents. The membrane potential for half maximal inactivation by standard Boltzman fitting (red line) was -40.1 mV with a slope of 4.7. (E) Recovery from fast inactivation utilizing a paired pulse protocol in the same cell as C. The single exponential time constant for recovery of inactivation was 1.7 ms (red line). (F) A different cell which elicited an overshooting action potential upon current injection under whole cell current clamp utilizing a potassium gluconate based intracellular solution. Inset: Response of the same cell under voltage clamp to a change in membrane potential from -80 mV to -10 mV elicited a peak current of 457 pA. Scale bars for inset: 5 ms, 0.2 nA.

Gary Johnson

1994-present-President, ICT

1993-1996-Conjugation Chemist, R&D Systems

19989-1993-Supervisor Protein Conjugation & ELISA Development Group, Solvay Animal Health

1986-1989-Immunologists, Biosciences Lab, 3M

1976-1986-Various Lab, U of MN

Gary’s Conatct Info:

• gary@immunochemistry.com
• 952-888-8788  

Inventing Better Ways to Measure Apoptosis 

This profile features another Scientist Entrepreneur. Dr Gary Johnson is the Founder and President of Immunochemistry Technologies LLC (ICT). His company manufactures kits that have the capabilities to quantitatively measure apoptosis effects. This is important to Neuromics, because these are core to many diseases of research interest to our customers. These range from Cancer where apoptosis detection can be used to to visualize the efficacy of tumor killing therapies to Neuroscience where apoptosis could be a root cause of many cognitive and neuro-muscular diseases.

I am excited about featuring Gary. I have been working with him and his team over the past 5 years. They have actively supported my company in providing Apoptosis Research Kits. The strength in our relationship is built on his company supplying best of breed reagents. The feedback I receive from users is overwhelmingly positive. In addition to these kits, ICT is also recoginized for their rock solid ELISA Buffers and Diluents.

It takes a unique blend of business and scientific acumen to build a company like ICT. So let’s start with Gary’s background and experience and then on to the specifics on his company and products and what sets ICT apart from competitors.

Gary’s Background

Gary’s began his career at the University of Minnesota in 1978 where he worked in a variety of labs. There he gained a wealth of experience and expertise in research techniqes. These included chromatography, immunoelectrophoresis, radiolabeling, mass spectrometry,  proton NMR spectroscopy and western blotting.

He leveraged his abilities and became more deeply involved in immunobiology. He  joined Dr. Harry Orr’s lab in 1981. There he used recombinant DNA techniques to study the class I genes of the major histocompatibility complex and he also supervised the tissue culture work. This provided the stepping stone to Dr. David Klein’s lab in 1984. There he studied the difference between diabetic and non-diabetic glomerular basement membrane proteoglycans in kidney disease. In order to do this research Gary developed in vivo or in vivo labeling techniques.

Gary then moved from University to commercial labs. We will see how his growing expertise morphed into the founding of ICT and why his broad knowledge and experise enabled a successful launch of the company.

From 1986 until founding ICT Gary worked at 3M, Solvay Animal Health and R&D Systems. Over his tenure, he worked as an Immunologist, Supervised an ELISA and Protein Purification and was a Conjugation Chemist. Having mastered a unique range of basic and commercial bio-research techniques, the evolution to Scientist-Entreprenuer was a natural next step.

In 1994, Dr. Brain Lee and Gary launched ICT. The company’s early success was in contract assay development. The revenue generated from these programs, has enabled ICT to manufacture and release a growing catalog of Apoptosis Detection Kits.

ICT’s Products and Capabilties

ICT’s provides proprietary probes for measuring apoptosis in vitro and in vivo. These probes are used by researchers  to detect caspases, cathepsins, serine proteases, cholinesterase enzymes, and assess mitochondrial health.Applications include: assessing the efficacy of chemotherapy, to quantifying  neurodegeneration, and early detectionof eye disease, to name a few.

Specific Products Include:

• FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits
• FLICA™ in vitro Caspase Kits
  • Fast!-Use Caspase kits to quantitate apoptosis via active caspases in whole, living cells. These kits do not use ELISA or any antibodies for detection
• FLISP™ Serine Protease Detection Kits
  • Measures chymotrypsin-like protease
    activation in whole living cells.
• Magic Red™ Real Time! Kits
  • Measures apoptosis in whole living, intact cells – no lysis required
• MitoPT™ Kits
• Quantitate mitochondrial functionality and apoptosis

Images: Normal (left) and keratoconus (right) corneal fibroblasts were labeled with Caspase 3 & 7 Assay Kit, green.

Pacing the Field

ICT is setting the pace in Apoptosis Detection by  recognizing and resolving issues inherent in competitive offerings. These include:

1. Difficulty permeating cells.
2. High background problems.
3. Does not bind to early stage apoptotic cells.
4. Not as sensitive as a cell permeant inhibitor probe.
5. Does not bind to all apoptotic tumor cells (Dicker, Cancer Biol. Ther., 2005. 9:1014-1017).
6. Binds positively to normal and healthy bone marrow derived cells (Dillon, J. of Immunol., 2001. 166:58-71).
7. Many in vitro protocols involve lysing the red blood cells before running flow cytometry, this method results in the binding of Annexin V to all of the cells in the sample (Tait, Blood, Cells, Molecules, and Diseases., 1999. 25:271-278).  The inversion of PS and cells containing large amounts of PS may not be related to apoptosis and this adds to the background issues.
8. Does not measure a process of apoptosis, but rather an effect of apoptosis.

Capabilities that will enable them strengthen their leadership position include:

1.  Uses a cell permeant probe that can easily penetrate tissues and cells.
2. Very sensitive.
3. Specific, no reported false positives.
4. It is a direct measurement of an intracellular process of apoptosis, detects only active caspases and caspase active cells are always apoptotic.
5. Passage through the blood-brain barrier has been demonstrated.
6. Passage through the blood-retinal barrier has been demonstrated.
7. No background problems when injected intravenously.
8. Detects very early through late stage apoptosis.

ICT is continuing to invest heavily in developing new capabilties. Gary highlighlighted some of the breakthroughs that are on the horizon. I plan on announcing these as they become public.Stay tuned.

It was further confirmed  in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’  i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement

Featuring Gary Johnson and his team at

The ability to accurately measure apoptosis processes is a core  research component for many of our customers and colleagues.  Neuromics has leveraged our growing partnership with Gary and ICT to meet the exacting requirements of  Researchers studying apoptosis.

I am excited to be featuring Gary and his company in our June News Behind the News. Gary and his team have

Polycaspase Apoptsis

proven to me time  and again the ability to deliver methods and kits that meet our customers’ needs.  I can count on the feedback to be positive and use to expand in user labs.

In the feature, I will provide details of Gary’s unique background. The path that lead him to founding ICT and the development of current capabilities. Most importantly, I will provide a glimpse of coming new methods and products. These could significantly improve the development of therapies for diseases that involve aptotosis.

Image: Jurkat cells dually stained with Hoechst and Polycaspase Assay Kit, green-FAM-VAD-FMK. Caspase activity is revealed by green fluorescence in cell #2, indicating that only this cell is apoptotic. Cell #1 is also dying (scattered blue), but is not apoptotic because it is not green. Cell #3 is healthy (concentrated blue nucleus).

I recently received impressive data and protocols from Emily Mcmains, a lab member.

Please note the excellent image of the cells 1 week after culturing and images taken after 4 days.

About Dr. Evanna Gleason Dr. Evanna Gleason Background: 1996-Current-Associate Professor, LSU 1993-1996-Post Doc, UC San Diego 1991-1992-Post Doc, UC Davis-Wilson Lab 1990 Ph. D- UC San Diego 1984 Undergrad-ASU

How do neurons communicate across synapses? Finding answers to this is of central interest to many of our customers and colleagues. After all, it is the transmission of signals across synapses that collectively orchestrate our perceptions.

Abnormal transmission is at the root of many neuro-disorders that plague society. Research in the cell and molecular biology of synapse transmission is a piece of the puzzle in discovering cures.

This leads to why I am honored to feature Dr. Evanna Gleason and her work on how Retinal Neurons Communicate. She and her team focus on how retinal synapses are specialized to transmit visual information. Her work adds to the body of understanding of the processes that enable us to see.

Beginnings

Assembling the pieces of Evanna’s research begin with her graduate work in Dr. Martin Wilson’s lab at UC Davis. Here she developed the culturing techniques required to study transmission between isolated pairs of amacrine cells. These techniques enabled the lab to study the firing of individual neurons and created the platform for her current research Here are related publications:

E Gleason, S Borges and M Wilson. Synaptic transmission between pairs of retinal amacrine cells in culture. Journal of Neuroscience, Vol 13, 2359-2370, Copyright © 1993 by Society for Neuroscience.

Gleason E., Borges S., Wilson M. Control of transmitter release from retinal amacrine cells by Ca2+ influx and efflux. Neuron 1994.  Nov;13(5):1109-17.

More on Amacrine Cells-Amacrine cells operate at the inner plexiform layer (IPL), the second synaptic retinal layer where bipolar cells and retinal ganglion cells synapse. There are about 40 different types of amacrine cells, most lacking axons. Like horizontal cells, amacrine cells work laterally affecting the output from bipolar cells, however, their tasks are often more specialized. Each type of amacrine cell connects with a particular type of bipolar cell, and generally has a particular type of neurotransmitter. One such population, AII, ‘piggybacks’ rod bipolar cells onto the cone bipolar circuitry. It connects rod bipolar cell output with cone bipolar cell input, and from there the signal can travel to the respective ganglion cells.They are classified by the width of their field of connection, which layer(s) of the stratum in the IPL they are in, and by neurotransmitter type. Most are inhibitory using either GABA or glycine as neurotransmitters.

 Developmental Neurobiology 

Evanna did her post doc in Dr. Nick Spitzer’s lab at UC-San Diego. She studied the development of voltage-dependent ion channels and neurotransmitter receptors in the embryo. The focus in the lab was more on systems assembly and differentiation vs the study of synaptic transmission between individual neurons.

Although an interesting sidetrack, Evanna shared with me that her passion is the study of synaptic transmission in retinal neurons. This bring us to her current work.

From San Diego to Baton Rouge

I became acquainted with Evanna in a phone follow up concerning use of our E18 Primary Rat Hippocampal Neurons. This conversation proved enlightening as she provided specific insight on what she did with the cultures. Growing healthy an robust cultures was the easy part.

Figures: Higher concentrations of NO promote a positive shift in E_GABA. A and B, top traces: raw data from ruptured-patch voltage-clamp recordings of GABA-gated currents from a representative cell before and after NO application. GABA pulses (20 µM) were 300 ms in duration and are indicated by horizontal bars. A: whole cell, voltage-clamp recordings (Cs⁺-A internal and TEA-A external) of GABA-gated currents reveal that higher concentrations of NO induce a transient, several-fold enhancement of GABA-gated currents. *, NO-dependent current observed prior to the 2nd GABA application. B: same experiment as in A, using air-exposed NO solution. Raw data in A and B are from same cell. Scale bars are 150 pA, 1 s. C: amacrine cell is held at the predicted E_GABA. GABA is applied for 300 ms during each trace. No GABA-gated currents are observed until application of NO. *, NO-dependent current. Scale bars are 25 pA, 5 s. D: voltage ramps in GABA were delivered before and after addition of NO. Leak-subtracted currents reveal a shift in E_GABA after NO application (gray trace). Inset: subtraction of the NO-induced shift in reversal potential reveals an increase in the slope of the GABA-gated current-voltage relationship after NO injection (gray trace). Scale bars are 100 pA, 20 mV. E: mean E_GABA values are plotted over time. F: representative GABA-gated currents from voltage ramps delivered after a 11-min treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 2 µM). Black trace, before NO injection; Gray trace, after NO injection. G: ODQ did not block the NO-induced shift in E_GABA (P = 0.83, n = 5).

As I learned from from this and my interview with Evanna: she and her team are assembling a clearer picture of the relationship between NO, Cl^-  and what is happing at  GABAergic synapses.

I plan to keep my eyes on how the puzzle grows and communicate the discoveries that bring the picture into clearer focus.  We will specifically be focused on the impact of Evanna’s research contributions to the overall understanding. of  how messages are communicated in the CNS and PNS.

Evanna indicated to me the potential of her using siRNA to do gene expression analysis.  As outlined in previous News Behind the Neuroscience News postings, this is near and dear to me. The ability to switch on and off genes involved in transmission will undoubtedly enhance the platform and drive new discoveries. 

I am pleased to be featuring Dr. Evanna Gleason.  I selected her because She and her team’s research is a basic component of most areas  Neuroscience Research including pain, neurodegeneration, vision, TBI, SCI, drug addiction, neuro-disorders and more…This base component is how neurons communicate with other cells at synapses. 

She focuses on synaptic transmission in the vertebrate retina. Retinal neurons have distinctive anatomical and physiological properties that suggest they employ unique synaptic mechanisms. The long term objective her research is to understand how retinal synapses are specialized to transmit visual information.

More to come in March!

About Dr. Laura Bohn  Dr. Laura Bohn  Background: Spring 2009-Associate Professor (tenured) at The Scripps Research Institute, Department of Molecular Therapeutics, Jupiter, FL. 10/2007- Associate Professor (tenured), The Ohio State University College of Medicine, Departments of Pharmacology and Psychiatry, Program in Pharmacogenomics 8/2003-9/2007 Assistant Professor, The Ohio State University College of Medicine, 1/1999–8/2003 Post-Doc/Assistant Research Professor. Duke University Medical Center, Department of Cell Biology. Durham, NC.

My company’s foundation is built on serving pain researchers. As a result, I have the good fortune of working with customers and collaborators who openly share the subtleties of their research and the future impact it could have on improving pain therapies.

Pain is complex. Today, pain therapies often fall short and are rife with unwelcome side effects. This undesrcores why I am pleased to feature Dr. Laura Bohn. She and her team are probing ways to improve  response effectiveness and reduce side effects.

 Beginnings

The story starts with Laura’s Post Doc work in Dr. Marc Caron’s lab at Duke University.  Marc in Collaboration with Dr. Dr. Robert Lefkowitz genetically engineered mice that lacked a protein switched called  “beta-arrestin 2.”  This switch is part of the opioid pathway that regulates how we perceive pain. The GPCR, muOpioid (mOR) is the primary target for narcotic pain killers, like morphine.

In her initial work, Laura found that morphine treated mice lacking the beta-arrestin2 switch swere able to tolerate  mild pain stimuli up to 3X longer than normal mice.  These mice had a higher level of sensitivity to morphine both in magnitude and duration. 

Bingo. This path for Laura’s excellent journey is now lit…understanding how the molecular regulation of G protein coupled receptors (GPCR) can translate to overall drug responsiveness in vivo.  Getting better response from lower dose is all good.

Current Work

As a researcher at Ohio State University, Laura and her team have continued to broaden and deepen their understanding of  GPCR signaling and beta-arrestin desensitivation (figure 1).

She is currently doing research with mice that have genetic deletions of GRKs (GRK3, GRK4, GRK5, and GRK6; heterozygotes for GRK2) and barrestin-2.

This expands the playing field. This expansion includes  studying other GCPR related pathways. Serotonin 2A receptors (5-HT2ARs), for example, are molecular targets for drug-induced hallucinations:

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn. Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo. Published online on January 14, 2008, 10.1073/pnas.0708862105.

The conclusion: 5-HT2AR–β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Future Considerations

I look for Laura and her team to continue the quest of doing more for less when it comes to novel pain and other therapies. Further success would provide the foundation for the development of therapies that would require less dosing, better response and reduced side effects.

Laura mentioned to me that further directions could involve the use of gene silencing tools like siRNA. The effects of silencing GPCR-beta-Arrestin receptors in-vivo would be an important study as it would enable she and her team to study  impact of  desensitivation on the repsonse to morphine and other drugs by normal mice.

Dr. Laura Bohn

January’s Story: Decoupling GPCR Pain Therapies from Destructive Side Effects. 

We are pleased to have Dr. Laura Bohn as our “coming soon” featured researcher. 

She caught my attention when she referenced one of our  Opioid Receptor Antibodies in the publication: C. E. Groer, K. Tidgewell, R. A. Moyer, W. W. Harding, R. B. Rothman, T. E. Prisinzano, and L. M. Bohn. An Opioid Agonist that Does Not Induce µ-Opioid Receptor—Arrestin Interactions or Receptor Internalization. DOI: 10.1124/mol.106.028258.

I am looking forward to drilling into the specifics of this important research.

Here they isolate, and maintain in culture, neural progenitors demonstrating properties of these neural epithelial cells from WA09 human embryonic stem cells (hESCs):

D.W. Machacek, S. K. Dhara, C. Sturkie, K. Hasneen, D. Carter, L. Murrah Hanson, P.R. MacLeish, M. Benveniste, S.L. Stice. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELL DERIVED NEURAL PROGENITORS INTO FUNCTIONALLY RESPONSIVE POPULATIONS IN THE ABSENCE OF EXOGENOUS EGF.