Protocol developed to harvest mouse cell lines for melanoma research

Dartmouth researchers have developed a protocol that permits cells harvested from melanoma tumors in mice to grow readily in cell culture. Their findings were published in an article, Multiple murine BRafV600E melanoma cell lines with sensitivity to PLX4032, in the January 25, 2014 issue of Pigment Cell & Melanoma Research.”We anticipate that these cell lines will be extremely useful to many investigators who use mouse melanoma as a model system,” said Constance E. Brinckerhoff, PhD, professor of Medicine and of Biochemistry at the Geisel School of Medicine at Dartmouth College and a member of the Norris Cotton Cancer Center (NCCC) Mechanism Research Program.There is a lack of mouse cell lines that harbor the BRAF mutation that is so prevalent in human melanomas, and the cell lines that are available grow slowly in culture and are not representative of human melanoma cell lines. Detailed experiments on molecular mechanisms controlling mouse cell line behavior have been difficult because the currently available mouse cell lines do not grow well in culture.The Geisel researchers are the first to have developed a protocol that permits mouse melanoma cells to be harvested from tumors in the mice and to grow readily in cell culture. Importantly, these cell lines are genetically compatible with a strain of mice that are immunologically competent, while human cells need to be placed into immunologically weakened mice in order to grow. Thus, the ability to study these mouse melanoma cell lines both in culture and in mice with an intact immune system is an experimental advantage.Story Source:The above story is based on materials provided by Norris Cotton Cancer CenterDartmouth-Hitchcock Medical Center. Note: Materials may be edited for content and length.

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Scientists ‘spike’ stem cells to generate myelin

Aug. 28, 2013 — Stem cell technology has long offered the hope of regenerating tissue to repair broken or damaged neural tissue. Findings from a team of UC Davis investigators have brought this dream a step closer by developing a method to generate functioning brain cells that produce myelin — a fatty, insulating sheath essential to normal neural conduction.”Our findings represent an important conceptual advance in stem cell research,” said Wenbin Deng, principal investigator of the study and associate professor at the UC Davis Department of Biochemistry and Molecular Medicine. “We have bioengineered the first generation of myelin-producing cells with superior regenerative capacity.”The brain is made up predominantly of two cell types: neurons and glial cells. Neurons are regarded as responsible for thought and sensation. Glial cells surround, support and communicate with neurons, helping neurons process and transmit information using electrical and chemical signals. One type of glial cell — the oligodendrocyte — produces a sheath called myelin that provides support and insulation to neurons. Myelin, which has been compared to insulation around electrical wires that helps to prevent short circuits, is essential for normal neural conduction and brain function; well-recognized conditions involving defective myelin development or myelin loss include multiple sclerosis and leukodystrophies.In this study, the UC Davis team first developed a novel protocol to efficiently induce embryonic stem cells (ESCs) to differentiate into oligodendroglial progenitor cells (OPCs), early cells that normally develop into oligodendrocytes. Although this has been successfully done by other researchers, the UC Davis method results in a purer population of OPCs, according to Deng, with fewer other cell types arising from their technique.They next compared electrophysiological properties of the derived OPCs to naturally occurring OPCs. They found that unlike natural OPCs, the ESC-derived OPCs lacked sodium ion channels in their cell membranes, making them unable to generate spikes when electrically stimulated. …

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Gene regulator is key to healthy retinal development and good vision in adulthood

Aug. 8, 2013 — Scientists are developing a clearer picture of how visual systems develop in mammals. The findings offer important clues to the origin of retinal disorders later in life.In research published this week in the Journal of Neuroscience, University at Buffalo scientists and colleagues focused on a particular protein, called a transcription factor, that regulates gene activity necessary for the development of one type of retinal neuron, the horizontal cells.Horizontal cells process visual information by integrating and regulating input from rod and cone photoreceptors, which allow eyes to adjust to see well in both bright and dim light conditions.”We have found that activation of the transcription factor named Onecut1 is essential for the formation of horizontal cells,” explains Xiuqian Mu, PhD, assistant professor in the departments of Ophthalmology and Biochemistry in the UB School of Medicine and Biomedical Sciences.The researchers came to this conclusion after creating mice that lacked Onecut1. In these knockout mice, the number of horizontal cells was 80 percent lower than in normal mice. The researchers were surprised to find that the removal of Onecut1 also had an impact on photoreceptor cells, the rods and cones that absorb light in the retina and convert that energy to an electrical impulse eventually conveyed to the brain.During development, Mu explains, the removal of Onecut1 only appeared to impact the horizontal cells. However, by the time these mice reached adulthood, around 8 months old, the level of photoreceptor cells in these knockout mice was less than half the normal level.”Because degradation of photoreceptors is believed to be a major factor in retinal diseases, such as retinitis pigmentosa and Leber’s congenital amaurosis, this finding, that horizontal cells are necessary for the normal survival of photoreceptor cells, is novel and significant,” says Mu. “Many retinal diseases are manifested by the degeneration of photoreceptor cells.”This finding was unexpected, Mu explains, because most investigations into the degeneration of photoreceptor cells have involved genes that directly affect photoreceptor cell development.”People haven’t been looking at horizontal cells,” he says. “We didn’t think that they’d be involved in photoreceptor cell degradation.”With this finding, we have discovered that retinal horizontal cells are required for maintaining the integrity of the retina and that their deficiency can lead to retinal degradation,” explains Mu.He notes that in most cases where photoreceptor cells die, it’s because they are somehow defective.”But in this case, the photoreceptor cells are fine in the beginning, so the death of the photoreceptor cells is a secondary affair that is somehow driven by the deficiency in horizontal cells,” he says.UB co-author Steven J. Fliesler, PhD, Meyer H. Riwchun Endowed Chair Professor, vice-chair and director of research in the Department of Ophthalmology and professor in the Department of Biochemistry, notes that this finding could open up a new area of study.”One scenario we have speculated upon is that there are important supportive interactions between horizontal cells and photoreceptors that are required to maintain photoreceptor function and viability,” Fliesler says. …

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The temperature tastes just right

Aug. 7, 2013 — Call it the Goldilocks Principle — animals can survive and reproduce only if the temperature is just right. Too hot and they will overheat. Too cold and they will freeze.To stay in their comfort zone, animals have evolved very sensitive temperature sensors to detect the relatively narrow margin in which they can survive. Until recently, scientists knew little about how these sensors operated.Now, a team of Brandeis University scientists has discovered a previously unknown molecular temperature sensor in fruit flies belonging to a protein family responsible for sensing tastes and smells. These types of sensors are present in disease-spreading insects like mosquitoes and tsetse flies and may help scientists better understand how insects target warm-blooded prey — like humans — and spread disease.The discovery is published in today’s advance online edition of the journal Nature.Biting insects, such as mosquitoes, are attracted to carbon dioxide and heat. Notice how mosquitoes always seem to bite where there is the most blood? That is because those areas are the warmest, says Paul Garrity, a professor of biology in the National Center for Behavioral Genomics at Brandeis who co-authored the paper.”If you can find a mosquito’s temperature receptor, you can potentially produce a more effective repellent or trap,” Garrity says. “The discovery of this new temperature receptor in the fruit fly gives scientists an idea of where to look for similar receptors in the mosquito and in other insects.”Professor of Biology Leslie Griffith and Associate Professor of Biochemistry Douglas Theobald assisted with the research, which was led by postdoctoral fellows Lina Ni and Peter Bronk.The newly discovered sensor belongs to a family of proteins, called gustatory receptors, that have been studied for over a decade but never linked to thermosensation, Garrity says. In previous studies, other gustatory receptors have been found to allow insects to smell carbon dioxide and to taste sugar and bitter chemicals like caffeine.But in fruit flies, one type of gustatory receptor senses heat rather than smell or taste. …

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Burnt sugar derivative reduces muscle wasting in fly and mouse muscular dystrophy

Aug. 1, 2013 — A trace substance in caramelized sugar, when purified and given in appropriate doses, improves muscle regeneration in a mouse model of Duchenne muscular dystrophy. The findings are published today (Aug. 1) in the journal Skeletal Muscle.Morayma Reyes, professor of pathology and laboratory medicine, and Hannele Ruohola-Baker, professor of biochemistry and associate director of the Institute for Stem Cell and Regenerative Medicine, headed the University of Washington team that made the discovery. The first authors of the paper were Nicholas Ieronimakis, UW Department of Pathology; and Mario Pantoja, UW Department of Biochemistry.They explained that the mice in their study, like boys with the gender-linked inherited disorder, are missing the gene that produces dystrophin, a muscle-repair protein. Neither the mice nor the affected boys can replace enough of their routinely lost muscle cells. In people, muscle weakness begins when the boys are toddlers, and progresses until, as teens, they can no longer walk unaided. During early adulthood, their heart and respiratory muscles weaken. Even with ventilators to assist breathing, death usually ensues before age 30. No cure or satisfactory treatment is available. …

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Biochemists identify protease substrates important for bacterial growth and development

June 27, 2013 — Reporting this month in Molecular Microbiology, Peter Chien and colleagues at the University of Massachusetts Amherst describe using a combination of biochemistry and mass spectrometry to “trap” scores of new candidate substrates of the protease ClpXP to reveal how protein degradation is critical to cell cycle progression and bacterial development. The new understanding could lead to identifying new antibiotic targets.As Chien (pronounced Chen) explains, to carry out fundamental life processes such as growing and dividing, cells must orchestrate, in time and location, the production and degradation of hundreds of protein substrates. Even in simple bacteria, protein degradation is critical for making sure these organisms can grow and respond to their environment properly.Scientists have known that a group of protein machines called energy-dependent proteases are responsible for the majority of this degradation, but what targets these machines recognize and how they do it has been unknown in many cases.With the new series of experiments in the model bacteria Caulobacter crescentus in the Chien biochemistry and molecular biology laboratory, much more is now understood, he says. “We first generated a protease mutant that could recognize but not destroy its targets, acting as a ‘trap’ for protease substrates. After purifying this trap from living cells, we used mass spectrometry to identify proteins that were caught, finding over a hundred new candidate substrates. These targets covered all aspects of bacterial growth, including DNA replication, transcription and cytoskeletal changes.”Next, they focused on one of these new targets in detail, a protein called TacA. Caulobacter grow by making two different cell types every time they divide. TacA is responsible for making sure that one of these cell types forms properly.”We used biochemistry and highly purified proteins to identify what parts of TacA were important for degradation by the ClpXP protease,” Chien says. “We then made mutants of TacA that could not be degraded and found that when we expressed them in bacteria, these cells failed to properly develop into the correct cell types. Because developmental changes are essential for pathogenic bacteria to invade their host, these insights could potentially identify new antibiotic targets.”The work was funded by a grant from the National Institute of General Medical Sciences at the National Institutes of Health and by UMass Amherst.

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