Wild Types has moved!

March 20, 2014 § Leave a comment

To a new URL: http://wildtypes.asbmb.org/

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Finding what makes biofilms hard to defeat in lung infections

March 10, 2014 § 2 Comments

Electron micrograph of a P. aeruginosa biofilm. Image provided by Cezar Khursigara.

Electron micrograph of a P. aeruginosa biofilm. Image provided by Cezar Khursigara.

Cystic fibrosis patients often combat lung infections. At the late stage of the infections, a bacterium called Pseudomonas aeruginosa becomes the persistent menace, forming a structure known as a biofilm. Bacterial biofilms are stubborn, tough structures that resist antibiotics and other means of removal. These structures also afflict AIDS and burn patients. In a paper recently published in Molecular & Cellular Proteomics, researchers have examined the entire protein content of P. aeruginosa biofilms. Their aim is to identify the molecular pathways that are critical to biofilm formation and maintenance, processes that are not well understood.

Cezar Khursigara at the University of Guelph in Canada, who spearheaded the work, says that he and one of his postdoctoral fellows, Amber Park, “wanted to examine the proteome dynamics of P. aeruginosa cells as they transitioned from a planktonic, free-living state to a biofilm lifestyle. This transition is thought to be similar to the one P. aeruginosa makes in the lungs of patients with CF, when pulmonary infections eventually move from an acute to chronic phase. “

The investigators compared the different protein expressions over the time in P. aeruginosa cells as they developed through the two different lifestyles. They discovered that there were significant shifts in protein expression as the bacteria switched from the planktonic state to the biofilm state. In particular, they saw differences in expression of proteins involved in iron acquisition and a downregulation of key antimicrobial targets.

“Most surprisingly, however, was the dramatic increase is cellular adhesins that were identified only in the later time points and were completely absent in the earlier planktonic samples,” says Khursigara. “We feel these may play a role in shaping biofilm architecture and, as a result, may contribute to the resilience of these communities.”

The investigators are now working on P. aeruginosa isolated from patients and developing experimental conditions that better mimic those in the lung. They are also plunging deeper into the pathways they have identified to see if they can pinpoint particular proteins that may be suitable as drug targets. Khursigara notes, “With the number of proteins identified, we will be busy for a while.”

Breast cancer gene involved in skeletal muscle energy metabolism

March 5, 2014 § Leave a comment

BRCA1 appears to play an important role in skeletal muscle. Image from http://phil.cdc.gov/phil/details.asp

BRCA1 appears to play an important role in skeletal muscle. Image from http://phil.cdc.gov/phil/details.asp

The BRCA1 gene, which is officially known as the breast cancer 1, early onset, gene, is well-known to be expressed in breast tissue. People who have particular mutations in this tumor suppressor gene are at increased risk of developing certain types of breast cancer. But in work just published in the Journal of Lipid Research, investigators demonstrated the BRCA1 gene also is expressed in skeletal muscle. Espen Spangenburg at the University of Maryland, the senior author on the paper, says that the work indicates that BRCA1’s influence “extends beyond just breast cells.”

Using cells taken from mice and humans, Spangenburg’s team demonstrated that there were multiple isoforms of BRCA1 in skeletal muscle.  They then showed that when mice underwent bouts of intense exercise, there were more interactions between BRCA1 and the phosphorylated form of acetyl CoA carboxylase, a critical regulator of lipid metabolism.

When the investigators reduced the BRCA1 content in human skeletal muscle cells in culture, the mitochondria consumed less oxygen. There was also more lipid stored inside cells, and the amount of insulin signaling dropped.

Taken together, the data suggest that BRCA1 is important in regulating energy metabolism in skeletal muscle. Furthermore, the work highlights that this gene plays a role in tissues beyond those involved in reproduction. Spangenburg says, “This is particularly important when one considers the number of known genetic mutations that develop in the BRCA1 gene. We need to consider how these mutations may affect skeletal muscle function.”

Method to visualize RNA molecules in individual cells

February 27, 2014 § Leave a comment

To develop fluorescent in situ sequencing, scientists first fix in place thousands of RNAs --  including working copies of genes called messenger RNAs -- in cells, tissues, organs or  embryos. Here, RNAs are labeled red in a mouse brain (left) and green in a mouse embryo  (right).  [Image courtesy of Wyss Institute and Harvard Medical School]

To develop fluorescent in situ sequencing, scientists first fix in place thousands of RNAs —
including working copies of genes called messenger RNAs — in cells, tissues, organs or
embryos. Here, RNAs are labeled red in a mouse brain (left) and green in a mouse embryo
(right).
[Image courtesy of Wyss Institute and Harvard Medical School]

Knowing where gene expression happens inside single cells is informational gold to scientists. In a paper just out in Science, researchers describe a technique that allows them to pinpoint where RNA molecules are located in individual cells.

Having a map of where RNA molecules are within single cells will help scientists better understand the dynamics of gene expression, which can differ significantly between cells. The map also can reveal how single-cell dynamics change under different physiological conditions, such as development and growth, or during disease processes, such as in various cancers.

The method, described by a team led by Je Hyuk Lee and George Church at Harvard Medical School, is called fluorescent in situ RNA sequencing, or FISSEQ.  Church says that for the method to be successful the investigators knew that they had to be able to sequence individual RNAs as well as hold the material steady and still at their locations.

In the current work, the researchers developed a way to cross-link amplified molecules of cDNA generated from the RNA transcripts into a highly stable matrix inside a cell. They imaged those cross-linked molecules using fluorescence microscopy and were able to tell where active gene expression was happening in the various parts of the cell.

As proof of concept, Lee, Church and colleagues obtained reads of 30 bases from more than 8,000 genes in human primary fibroblasts. They showed that they could track changes in expression in those cells with a simulated wound-healing assay.

The researchers have applied FISSEQ to other cell types, including mouse embryos, mouse brain sections, whole-mount Drosophila embryos and human stem cells. “It seems to work on every situation that we’ve tried,” says Church.

Church says because the FISSEQ seems to work in most sample types, “clinical pathology specimens should be directly adaptable to FISSEQ. We can learn if previously similar-looking cells are actually molecularly significantly different.” He adds that the data could reveal the heterogeneity in tumors, inflammatory processes and growth of blood vessels

At the moment, the researchers are applying FISSEQ to the Brain Research through Advancing Innovative Neurotechnologies, or BRAIN, Initiative that was rolled out by the Obama administration last year. Church says they are collecting “diverse types of data — RNA, cell connections, developmental lineages and time records of the signals going through those connections — all integrated into a single brain sample with related behavioral data.”

Splitting up kinases

February 26, 2014 § Leave a comment

A ligand-inducible split-kinase is activated by a small molecule. Image by Karla Camacho-Soto.

A ligand-inducible split-kinase is activated by a small molecule. Image by Karla Camacho-Soto.

Researchers have figured out a way to bend kinases at their will. In a paper recently published in the Journal of the American Chemical Society, Indraneel Ghosh and colleagues at the University of Arizona describe engineering these enzymes, which are critical for signaling pathways, so that they can be controlled by researchers. These engineered kinases should “allow for a more precise understanding of signaling cascades that are currently unavailable,” says Ghosh.

Scientists have been interested in manipulating enzymes with exquisite precision because the manipulation helps them to understand how these molecular machines work. Kinases also represent a lucrative class for drug targets because many of them have been identified to be involved in disease conditions, such as cancer. Knowing how kinases work in detail helps to develop highly targeted drugs.

Ghosh and colleagues demonstrated that one way to turn kinases on and off at will was to split the enzymes into two. They then connected the two parts of the enzyme with a special linker. When given a specific small molecule, such as the immunosuppressant rapamycin, two parts of the split enzyme came together and became activated like their normal counterparts. Because these enzymes could be activated with a small molecule, Ghosh and colleagues called them “ligand inducible split-kinases.”

The researchers showed that the approach could be used on four different kinases, suggesting that the method is broadly applicable. Ghosh says the long-term goal of the project is to have multiple ligand inducible split-kinases within a cell so that researchers can understand how these enzymes work relative to one another in the complex network of signaling pathways.

Broader look at proteins involved in learning and memory

February 19, 2014 § Leave a comment

Schematic shows a protein array and parts of signaling pathways relevant to learning and memory. The yellow boxes indicate proteins that changed in level with normal learning. Image provided by Katheleen Gardiner.

Schematic shows a protein array and parts of signaling pathways relevant to learning and memory. The yellow boxes indicate proteins that changed in level with normal learning. Image provided by Katheleen Gardiner.

Learning is complicated business, but typical research studies into the molecular basis of learning and memory measure only one or a few proteins. In a study just reported in the journal Molecular & Cellular Proteomics, researchers cast a wider net and looked at 80 proteins in the brain of mice. By looking at more proteins, the study leader’s Katheleen Gardiner at the University of Colorado says researchers can get a better appreciation of “the greater complexity of molecular events underlying learning and memory, how components of a single pathway change in concert and how many pathways and processes respond.”

Gardiner’s research focus is on Down syndrome, two characteristics of which are that patients suffer from some level of intellectual disability and eventually develop Alzheimer’s disease. Gardiner’s group aims to find drugs that can lessen the learning disability. But, in order to do that, researchers need to better understand the molecular events associated with learning, memory, and neurodegeneration.

To get a grasp of the proteins involved in a particular learning process, the investigators studied context fear conditioning in mice. In this type of experiment, mice are put in a new cage and given a small electrical shock. Researchers can tell when a mouse has learned to be fearful of the same cage when the mouse freezes when put back in the cage. This approach “has the advantage that it requires only a single trial, lasting less than five minutes, for mice to learn,” explains Gardiner. “This means that we have a clear window in time where we know molecular events associated with successful learning occur.”

Context fear conditioning demands that the hippocampus, a region of the brain important for memory formation, be functional. The hippocampus is also a part of the brain that degenerates in Alzheimer’s disease.

The investigators gave the mice a drug called memantine, which is used to treat moderate to severe cases of Alzheimer’s disease. The drug has been shown to correct for learning impairment in a mouse model of Down syndrome.

Gardiner’s group used proteins arrays to see how protein expression changed in the brains of mice that underwent context fear conditioning and were given memantine compared with control mice. They found levels of 37 proteins changed in the nuclear fraction of hippocampus. Abnormalities in 13 proteins had been reported in brains of Alzheimer’s patients. “One surprise was that many proteins that increased in level with normal learning also increased, although not as much, with treatment with memantine alone,” says Gardiner. “Memantine induces responses in a substantial number of proteins that we measured, and it does this without impairing or enhancing learning. This indicates that there is considerable flexibility in the timing and extent of protein responses that still result in successful learning.”

In particular, Gardiner’s group identified the MAPK and MTOR pathways to be affected in their experiments, as well as subunits of glutamate receptors and the NOTCH pathway modulator called NUMB. NUMB is known to be essential for some aspects of brain development.

Gardiner says her group is now looking at data from a similar experiment done with a mouse model of Down syndrome. Those mice were unsuccessful with context fear conditioning, but they did as well as wild-type mice when they were treated with memantine.

Figuring out the target for Lorenzo’s oil

February 7, 2014 § Leave a comment

Researchers have figured out how Lorenzo’s oil works. According to a team led by Akio Kihara and Takayuki Sassa at Hokkaido University in Japan, the mixture of oils made famous in the 1992 movie “Lorenzo’s Oil,” inhibits an enzyme that is critical for making a specific type of fatty acid chains.

Lorenzo’s oil, a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, is used to treat a peroxisomal disorder called X-linked adrenoleukodystrophy (X-ALD). In this disease, very-long-chain saturated fatty acids don’t get degraded. They accumulate in the peroxisomes and clog them up. The defect goes on to wreck the sheath around neurons, leading to poor muscle coordination, vision loss, aggressive behavior and other symptoms. Lorenzo’s oil, with its fatty acid chains of 18 and 22 carbons, somehow normalizes the levels of saturated fatty acid chains with 24 and 26 carbons in the blood of X-ALD patients.

However, researchers have not figured out precisely how this mixture actually works at the biochemical level. In a paper just out in the Journal of Lipid Research, Kihara and colleagues demonstrated that Lorenzo’s oil targets an enzyme called ELOVL1, which makes fatty acids with more than 20 carbons.

Kihara’s group members had done a lot of work on ELOVL1 so they knew it was the main enzyme for making these very-long-chain fatty acids. “We thought it possible that Lorenzo’s oil may prevent saturated very-long-chain fatty acids from accumulating by inhibiting their synthesis through ELOVL1,” says Kihara.

The investigators already had a way to quantitatively track the activity of ELOVL1. They looked to see what effect the compounds in Lorenzo’s oil had on the enzyme. They anticipated, given ELOVL1’s role in making very-long-chain fatty acids, that Lorenzo’s oil inhibited ELOVL1. They were right. When they tested various ratios of the fatty acids in Lorenzo’s oil, oleic and erucic acids, they found the 4:1 mixture—the actual Lorenzo’s oil composition—was the most potent. The data from the various mixtures suggest that the two fatty acids in Lorenzo’s oil cooperate to inhibit ELOVL1 in places away from the substrate binding site.

At a cellular level, the investigators noted that the 4:1 mixture lowered the level of sphingomyelin made from a saturated very-long-chain fatty acid and raised the level of sphingomyelin with a monounsaturated very-long-chain fatty acid. This result may explain why Lorenzo’s oil can help reduce the risk of developing X-ALD in asymptomatic patients (who are always boys)—sphingomyelin is an important component of the sheath that goes around neurons.

Because X-ALD is caused by impaired degradation of very-long-chain fatty acids, restoring the degradation process is the obvious strategy for a treatment. But Kihara says their work suggests that stopping the very-long-chain fatty acids from being made could be an alternative. As the oleic and erucic acids bind to ELOVL1 away from the substrate binding site, Kihara says the investigators think these two oils could be lead compounds for the development of specific inhibitors of ELOVL1 that don’t affect other enzymes involved in making very-long-chain fatty acids.

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