March 20, 2014 § Leave a comment
To a new URL: http://wildtypes.asbmb.org/
See you there!
March 10, 2014 § 2 Comments
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.”
March 5, 2014 § Leave a comment
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.”
February 27, 2014 § Leave a comment
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.”
February 26, 2014 § Leave a comment
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.
February 19, 2014 § Leave a comment
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.