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Morphoproteomics
Posted on July 9th, 2010 No commentsMorphoproteomics is a novel medical discipline that combines histopathology, molecular and cellular biology and protein biochemistry to define the protein circuitry in diseased cells. In this way it should be possible to identify specific target for customized therapeutic intervention. One preclinical study on prostate cancer overlaps data from phosphorylation analysis with phospho-specific probes and morphological evaluation of cellular compartmentalization. The identification of peculiar localization of certain phospho-analytes demonstrated the efficacy of this kind of personalized studies and their clinical utility. Another study demonstrated the feasibility of this approach in neck and head squamous carcinoma, that often has poor prognosis and high mortality rate. In this case, mTOR pathway was analyzed in terms of potential clinical significance of its activated components. Rapamycin is an inhibitor of this pathway. The inhibitory effects on cellular growth were directly correlated with mTOR activation, and a paint of the block was obtained by immuno-hystochemistry. On conclusion, morphoproteomics gives details about the signal transduction by using the combination of several techniques. This approach has been positively evaluated in certain disease, like cancer where an alteration in protein circuitry often causes the disease. Other pathological model would be available soon to validate this approach also in other disease.
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Challenging on transcriptome
Posted on July 2nd, 2010 No commentsmRNA translation is one of crucial process in cellular metabolism. Several approaches have been developed by cells to perfectly regulate the translation. microRNAs bind mRNA and determine its half life, while numerous proteins directly bind the mRNA and favor or avoid the interaction with ribosomes.
Studying the interaction between proteins and mRNAs is important to well understand whether alteration in these binding sites have any contribution to genetic disease that are caused by a loss of an RNA- binding protein such as fragile X mental retardation or familial amyotrophic lateral sclerosis. Scientists form the Rockefeller University described a method to isolate complexes protein- RNA. They used the UV radiation that are known to form covalent bonds between protein and mRNA. So, live cells are exposed to UV radiation, and an immunoprecipitation against the protein binder allows to identify the sequence involved in the binding. This technique called CLIP (PDF), cross linking and immunoprecipitation, can be applied also to high throughput experiments. To do this some improvement are necessary in order to separate the signal from background. The use of photo- activable ribonucleotides introduces into the RNA some non-toxic and efficient linkers. Indeed, a specific base change during reverse transcription, scoring for thymidine to cytidine in the sequenced cDNA allows to precisely map the interaction site. This is an important step to unravelling the gene regulation. -
Cellular role of chloride movement
Posted on June 18th, 2010 No commentsThe movement of ions inside or outside the cellular membrane is crucial to maintain pH homeostasis, the membrane potential and in some cases they may regulate also the entrance of drugs, glucose and so on. The chloride ion moves into the cells through the intracellular member of the chloride channels family. Chloride moves into the organelle lumen toward a positive electrical potential firstly generated by the activity of proton pump.
This movement destroys the electrical potential and allows vesicle acidification. In contrast, it has been recently hypostasized and demonstrated that cells exploit proton gradient to concentrate the chloride ions inside the endosomes and other vesicles. A similar activity has been described in plants to accumulate the nitrate. Animal models that present knock out CLC proteins show kidneys symptoms or osteopetrosis, like in human disorders. These genetic alterations uncouple the chloride channels from proton exchangers. Therefore, the main characteristic of chloride movement is the coupling to proton movement. In yeast, chloride- proton coupling is related to multicopper oxidase synthesis, and a problem at ion movement levels generates immature oxidases. In conclusion, ions movement is functional not only for hydrolytes balance, but also to metabolism and biogenesis. Next works will clarify better these processes. -
Embryonic stem cells in diabetes therapy
Posted on May 26th, 2010 No commentsBeta cells transplantation seems the most promising therapy for diabetes, because other current treatments loss their favorable advantage and can not avoid serious adverse effects, such as hyperglycemia. Embryonic stem cells are the ideal biological tool to achieve the complete restoring of pancreatic islet. Current research on diabetes is focused on the identification of a protocol to culture cells.
Indeed, embryonic stem cells must be differentiated in order to produce insulin. It has been recently published that high concentration of exendin 4 are able to induce insulin synthesis and differentiation to pancreatic cells. This strategy is a modification of the original protocol that requires the simultaneous administration of exedin 4, nicotinamide and activin b. Additional culturing in low glucose medium seems to further improve embryonic cells differentiation. Despite previous studies, Hu and coworkers (Acta Pharmacologica Sinica) obtained more insulin 1 and peptide C. This study gives great hope to patients that suffer for diabetes and can not have a benefit from traditional approaches. Embryonic stem cells must be further studied to improve culturing conditions in order to achieve cellular population useful for transplantation. -
Endocytosis
Posted on May 24th, 2010 No commentsEndocytosis is a process crucial for cellular life. Through endocytosis, extracellular components can be internalized and metabolized. One situation in which endocytosis is extremely important is the synaptic communication. Indeed, to finely tune the signal, endocytosis contributes to remove receptors from the cellular membrane and stop the signal. Several kind of endocytosis have been described; the common one is the clathrin dependent endocytosis, where coated pits arise from cellular membrane and move into the cytoplasm.
During this movement, the pH inside the pits lowers and allow the detachment of receptors and ligands, for instance. Proteins that remain into the membrane are recycled, the others are degraded by proteosoma. The network that regulates endocytosis is quite complex. Some signals derived from membrane receptors self that change their three- dimensional conformation and recluse clathrin. Otherwise, some intracellular signals allow endocytosis; actin filaments transport the pits to proteosoma. Endocytosis requires a lot of energy, derived from ATPase activity but allows at the same time to save membrane and recycle proteins. Rather than endocytosis, it has been described esocytosis and pynocytosis, that control efflux from cells and liquid internalization, respectively. All these processes are really interesting to study and a lot of open questions are waiting for a solution. -
Cellular transfection
Posted on May 17th, 2010 No commentsExperimental research uses modern and old cellular biology techniques for an high number of applications. For this reason, cellular biology shows great advances in last decades. Improvement in cell culture media allows propagating cell lines that was not possible to culture just few years ago. For instance, stem cells are now available and can be maintained in plate without loosing their stemness. Despite these advances in cell culturing, the method of transfection, most widely used, still is the calcium phosphate method. Indeed, either adherent and in suspension cells can be treated with this technique and can be successfully transfected.
The general protocol requires the direct addition of calcium phosphate DNA complex to cell medium, at least for six hours. Nevertheless, some modifications have been proposed in order to increase transfection. It could be possible to pre-treat cells with chloroquine, that is able to produce pores into cellular membranes. Otherwise, it’s also possible to shock cells with glycerol for less than two minutes. After glycerol treatment, it’s fundamental to accurately rinse cells with sterile PBS in order to completely remove the glycerol self. The calcium phosphate method is also inexpensive because all reagents can be produced in house, by using salts and powders usually present in research lab. -
Benefic effects of Thalidomide on vessel maturation
Posted on May 4th, 2010 No commentsThalidomide is an antiemetic drug sadly famous for its dangerous effects during pregnancy. This drug was not tested on right animal model and generated a long series of congenital malformations in babies born from mother that had used it. For several years thalidomide has been forgotten, up to date when it has been studied to treat leprosy and cancer. This month Nature Medicine publishes an interesting paper concerning thalidomide effects on vessel maturation. Scientists from the College de la Santè in Paris and the Curie Institute demonstrated that thalidomide contributed to reduce epistaxis in individuals affected by hereditary hemorrhagic telangiectasia.
This disease is characterized by vessel malformation that leads to frequent hemorrhages and nosebleeds, seriously altering the quality of life. Thalidomide reduced the frequency of nosebleed and increased hemoglobin level. Furthermore, in mice thalidomide induced vessel wall repairing. Molecular mechanism of this process seemed to involve PDGF-B that stimulated mural cells to the activation. Indeed, pharmacological blocking of PDGF signaling pathway reverted the positive effect of thalidomide. In summary, this paper shows a new application of an old drug. Pharmacological research may use this approach to identify new therapies by further investigating old drugs that have been previously approved and tested on humans. -
Challenge in PCR
Posted on April 22nd, 2010 No commentsPCR has become the most used technique in several field of molecular biology and diagnostic within few years since its invention. The majority of DNA fragment can be amplified without any problem; nevertheless, some regions into the genome are not so easily replicated, such as many regulatory domains, promoters, enhancers or other control elements. The main reason for all these difficulties is the high GC content that causes the formation of intramolecular stem loops during the initial cycles resulting in lack of amplification.
The slowdown PCR is a novel technique characterized by the use of slow ramping rates and an increased number of cycles at the same annealing temperature. Furthermore the addition of dc7GTP to normal dGTP in a ratio 1:3 maximally improves the reaction. Crucial step to do slow down PCR is to find an instrument –old- that uses slow ramp; in contrast modern instruments usually work at 5°C/sec to cool temperature. Then, also the choice of polymerase can contribute to obtain good results, this method may be limited only by the half- life of polymerase. However, Taq polymerase usually gives a positive amplification. In conclusion, the slow down PCR allows continuing important studies about gene expression regulation, by cloning DNA control sequence. -
A gift from sushi
Posted on April 14th, 2010 No commentsSeveral questions arise about genetically modified organisms and the possibility of gene transfer. Of course, everyone works in science knows that is not possible that gene containing in OGM can move into our genome or modify it in whatever manner. Otherwise, a recent article published in Nature journal shows a transfer of carbohydrate active enzyme from marine bacteria to gut bacteria. Authors started from the observation that Japanese population presented two enzymes able to metabolize agar and porphyran of red algae, while western population didn’t show any similar genes from metagenome analysis.
Seaweeds seemed to be the most likelihood vehicle for this transfer, especially because Japanese people eats an average of 14,2g per die of seaweeds. Furthermore, terrestrial plants don’t have similar enzymes. Few information were available in literature about substrate specificity of marine enzymes and their ability to digest agars and carrageen.
Scientists isolated five enzymes from a marine bacterial strain, frequently found on seaweed and expressed two of them in E.Coli –they analyzed only soluble proteins-. By analyzing several possible substrates, they demonstrated higher specificity versus natural polysaccharides extracted from various marine macrophytes and described a new class of glycoside hydrolases. To better explain the mechanism of action and compare it with traditional hydrolases, scientists solved the three-dimensional structure.
Structure-based alignment indicated that important residues for porphyran recognition were extremely conserved. Metagenomic data confirmed that no porphyranases were present in samples with terrestrial origin or open ocean origin, because seaweed is usually limited to coastal region.
In contrast, gut metagenome data from Japanese volunteers showed that these enzymes were present in human gut. Furthermore, in one family either the mother and her unweaned baby girl had a microbiota containing porphyrinase genes, porphyranolytic bacterial strains may be transmitted from mother to child. Altogether, these data indicated that porphyranases are specifically encountered only in Japanese gut and are probably absent in the microbiome of western people. The transfer from seaweed to human gut probably happened in recent time compared to the millions of years of gut microbiome evolution. Indeed, giving omnivorous human diet, other plant specific genes have been acquired, but these are present in all populations. This study offers an example of how our body is able to adapt itself to the microenvironment, not only form an enzymatic point of view, but also with genetic changes. The next challenging question is to understand which is the biological mechanism that allow the transfer and how it’s possible that only this kind of genetic transfer happens in human gut. Indeed, we continuously ingest non sterile food, but not in all cases gut microbiome modifications are noticed. We need to remember that even if transfer is quite recent, Japanese probably eat sushi and seaweeds since thousands of years, so possible genetic modifications in our gut microbiome will be recognized within thousands of years, and this is not our problem! -
Novel technique to diffract molecules
Posted on March 25th, 2010 No commentsAnkylography is a novel technique that is setting up to determine the three-dimensional structure of proteins or in general macromolecules. This technique is based on the opportunity to measure the diffraction pattern through a spherical detector.
A coherent x-ray beam is launched over a single particle and the diffraction records of scattered waves is registered on a curved surface. In this way, the three- structure of an object is encoded by the 2D spherical pattern and is solved in a single shot.
Ankylography is currently at the beginning, but several applications have been already imagined: for instance, it will be possible to study the enzymatic conformational changes in the real time of the reaction, thus making realistic the opportunity to develop drugs and inhibitors for each enzymatic state. Indeed, the biggest difference between ankylography and traditional x- ray diffraction is the capability in the first case to determine the structure of a single molecule and not an average of different conformations.
To do this, additional studies and developments are needed, from a new generation of powerful x- ray generators to algorithms to elaborate the huge amount of data derived from similar experiments. Fortunately, US and Europe governments are interested in ankylography and seem to invest money in it.
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