Archive for May, 2010

Event: Journal Club

Benoit Mouzon will be presenting “Blood-brain barrier breakdown and repair by Src after thrombin-induced injury” by Liu et al.

The journal club will start at 4:00pm on Friday (may 28th 2010) in the auditorium at Roskamp Institute.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

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Announcement

The Roskamp Institute Memory Center and Clinical Trials Division is pleased to announce the addition of Ms. Yahdinah Alvarez to the clinical team.  Ms. Alvarez is an experienced clinical trial coordinator with expertise in Alzheimer’s and Parkinson’s disease studies.  If you or your loved one is suffering with Alzheimer’s disease or Parkinson’s disease, please contact the clinic at (941) 256-8018 for information regarding possible treatment options.

The Roskamp Institute Memory Clinic and Clinical Trials Division provide a full range of services for individuals with Alzheimer’s disease including diagnostic work-up and follow-up treatment, neuropsychological examination, clinical trial opportunities, and memory screening.  For more information, please call (941) 256-8018.

News:Dr Crawford will be speaking at 2nd Annual Girls S.T.E.M. Summit

2nd Annual Science, Technology, Engineering & Math (S.T.E.M.) Summit at USF  Sarasota-Manatee on Saturday,  will start at 9 AM in May 22nd. Dr Crawford will give a speech about women in science.

For more information visit S.T.E.M. website.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

Alzheimer's beta-amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2

By Patel NS, Mathura VS, Bachmeier C, Beaulieu-Abdelahad D, Laporte V, Weeks O, Mullan M, Paris D.

Published in J Neurochem. 2010 Jan;112(1):66-76. Roskamp Institute, Sarasota, Florida, USA. npatel@scripps.edu

Abstract

Beta-amyloid peptides (Abeta) are the major constituents of senile plaques and cerebrovascular deposits in the brains of Alzheimer’s disease patients. We have shown previously that soluble forms of Abeta are anti-angiogenic both in vitro and in vivo. However, the mechanism of the anti-angiogenic activity of Abeta peptides is unclear. In this study, we examined the effects of Abeta1-42 on vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, which plays a key role in angiogenesis. Abeta inhibited VEGF-induced migration of endothelial cells, as well as VEGF-induced permeability of an in vitro model of the blood brain barrier. Consistently, exogenous VEGF dose-dependently antagonized the anti-angiogenic activity of Abeta in a capillary network assay. Abeta1-42 also blocked VEGF-induced tyrosine phosphorylation of VEGFR-2 in two types of primary endothelial cells, suggesting an antagonistic action of Abeta toward VEGFR-2 signaling in cells. Moreover, Abeta was able to directly interact with the extracellular domain of VEGFR-2 and to compete with the binding of VEGF to its receptor in a cell-free assay. Co-immunoprecipitation experiments confirmed that Abeta can bind VEGFR-2 both in vitro and in vivo. Altogether, our data suggest that Abeta acts as an antagonist of VEGFR-2 and provide a mechanism explaining the anti-angiogenic activity of Abeta peptides.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

Tech Tips, #2: Making a homemade frit for fused silica columns

By our own MS guru Jon Reed

A Thermo Engineer once told me that “Nano-LC is not for the faint-of-heart.” A 0.5µl bubble in a 10-port valve can make a mess of an experiment.  Incomplete proteolytic digestions can clog up your brand new $600 column and render it useless.  A few centimeters of post-column dead volume can cause significant peak broadening despite the fact that you have a $50,000 LC system.  The list of problems goes on and on.   This causes more than its share of heartache, which leads to this simple truth:

If the favorite pastime of proteomics researchers is bragging to one another about their latest and snazziest technological acquisitions, then the second favorite pastime is whining about how they don’t work.

Still, as time progresses, people chip away at old problems and find out how to do things better, faster, cheaper.  So this week, I’ll leave you with one less thing to cry about and describe HOW TO MAKE A BETTER HOMEMADE FRIT FOR FUSED SILICA COLUMNS.

I had originally learned to make frits by dipping a piece of fused silica into 75% KASIL and 25% DMF, however these frits were lengthy, un-reproducible, and lead to high back pressure and inconsistent chromatography.  That technique sucks.  Sorry, stinks.  No… wait… it sucks.

A new and improved protocol was first shared with me by Jennifer Busby and Valerie Cavett from Scripps.  They’re very smart, and you should read some of their papers. Go on… log on to Pubmed and get to reading.

It’s an adaption of earlier work by Maiolica et al (Proteomics 2005, 5, 3847–3850), and takes a whopping 2 minutes, and $0.25 of reagents from start to finish.  Well, that doesn’t include the ½ hour drying time, but if you’re saving that kind of time and money for a superior product, don’t complain!

OK, here goes…

  1. Make a Kasil/formamide mix (75/25) and use approximately 2 µL to wet a glass microfiber filter (GC/F, Whatman).
  2. Gently push and twist the end of a fused silica capillary onto the wetted filter.
  3. Dry the frit for 5 minutes at approximately 37ºC or at room temperature for approximately 20 minutes before packing.  Note: times given for a 75 µm capillary; larger diameters may need longer to dry before packing.

Note: different column IDs may require a 2nd glass fiber plug (do not wet this one) to ensure you don’t blow the frit loose.  To do this, first push the end of the fused silica tubing into a dry piece of filter and core out what you need, then repeat this on the wetted section of filter.  You may want to try this using 2 wetted cores, but that may lead to increased back pressure.  The only way to know is to try it out.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

Do You Or Someone You Care For Suffer From Alzheimer’s Disease?

ALZHEIMER’S DISEASE

A research study is being conducted in our area to evaluate an investigational medication for Alzheimer’s disease.

Qualified participants may receive:

– Study related medical care

– Study medication

– Compensation for time and travel

Please call now for more info:    Dr. Andrew Keegan

941-256-8018 ext. 353

The Roskamp Institute Memory Clinic and Clinical Trials Division provide a full range of services for individuals with Alzheimer’s disease including diagnostic work-up and follow-up treatment, neuropsychological examination, clinical trial opportunities, and memory screening.  For more information, please call (941) 256-8018.

Reduction of b-amyloid pathology by celastrol in a transgenic mouse model of Alzheimer’s disease

By Daniel Paris*, Nowell J Ganey, Vincent Laporte, Nikunj S Patel, David Beaulieu-Abdelahad, Corbin Bachmeier, Amelia March, Ghania Ait-Ghezala, Michael J Mullan published in Journal of Neuroinflammation 2010, 7:17

Abstract

Background: Ab deposits represent a neuropathological hallmark of Alzheimer’s disease (AD). Both soluble and insoluble Ab species are considered to be responsible for initiating the pathological cascade that eventually leads to AD. Therefore, the identification of therapeutic approaches that can lower Ab production or accumulation remains a priority. NFkB has been shown to regulate BACE-1 expression level, the rate limiting enzyme responsible for the production of Ab. We therefore explored whether the known NFkB inhibitor celastrol could represent a suitable compound for decreasing Ab production and accumulation in vivo.

Methods: The effect of celastrol on amyloid precursor protein (APP) processing, Ab production and NFkB

activation was investigated by western blotting and ELISAs using a cell line overexpressing APP. The impact of celastrol on brain Ab accumulation was tested in a transgenic mouse model of AD overexpressing the human APP695sw mutation and the presenilin-1 mutation M146L (Tg PS1/APPsw) by immunostaining and ELISAs. An acute treatment with celastrol was investigated by administering celastrol intraperitoneally at a dosage of 1 mg/Kg in 35 week-old Tg PS1/APPsw for 4 consecutive days. In addition, a chronic treatment (32 days) with celastrol was tested using a matrix-driven delivery pellet system implanted subcutaneously in 5 month-old Tg PS1/APPsw to ensure a continuous daily release of 2.5 mg/Kg of celastrol.

Results: In vitro, celastrol dose dependently prevented NFkB activation and inhibited BACE-1 expression. Celastrol potently inhibited Ab1-40 and Ab1-42 production by reducing the b-cleavage of APP, leading to decreased levels of APP-CTFb and APPsb. In vivo, celastrol appeared to reduce the levels of both soluble and insoluble Ab1-38, Ab1-40 and Ab1-42. In addition, a reduction in Ab plaque burden and microglial activation was observed in the brains of Tg PS1/APPsw following a chronic administration of celastrol.

Conclusions: Overall our data suggest that celastrol is a potent Ab lowering compound that acts as an indirect BACE-1 inhibitor possibly by regulating BACE-1 expression level via an NFkB dependent mechanism. Additional work is required to determine whether chronic administration of celastrol can be safely achieved with cognitive benefits in a transgenic mouse model of AD.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949