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Lipopolysaccharide induces the expression of T-like receptor 9
Posted on September 2nd, 2010 No commentsThe human bronchial epithelial cells have been recently used to investigate the expression of Toll like receptor 9, after the induction by lipopolysaccharide. This receptor has a transmembrane structure and signals though MAPK and IKK pathway, at last instance. It is involved in the innate immune response: indeed, it recognizes the molecules shared by pathogen, as different from the host ones and generates a response. Firstly, IL2 and IL18 are produced in antigen presenting cells, and these cytokines induce the maturation of naïve T cells. Then, pathogens are captured and internalized into the cells through phagocytosis or endocytosis. Lipopolysaccharide is usually expressed on bacterial surface and it’s reasonable to hypothesize that the interaction between LPS and human cells induces the expression of proteins involved in the innate immune response. The work published in the last number of Nature Preceding demonstrates and confirms this hypothesis. Scientists at the Department of Chemistry of Anna University (India) showed by RT-PCR and western blot analysis the induction of T- like receptor 9 after stimulation by LPS. This novel and innovative approach may be applied to study the regulation of other proteins, in order to better understand the mechanism that controls the innate response.
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Pfam database
Posted on August 30th, 2010 No commentsPfam is a novel database that contains conserved proteins and domains, usually employed in proteome analyses or sequencing classification. In this database, proteins are classified into two groups: Pfam-A families are manually annotated and inserted into the database after overcoming strictly threshold, while Pfam-B families are automatically created and clustered through similar sequence regions not matched with Pfam-A. A further rearrangement divides Pfam-A families in clans in respect to a hierarchical classification. The proteome coverage of Pfam database is different among species: information about bacteria are the most complete. Coverage is defined as sequence or amino acid coverage. In the first case, the proportion of sequence with a match with at least one of Pfam-A family describes the grade of completeness of the information. In the second case, is the proportion of amino acid belonging to the Pfam-A family that corresponds to the coverage. In order to grow and increase the information contained in the database, authors affiliated to the Wellcome Trust Sanger Institute, require the contribution of all scientists working on proteome. They are looking for new alignments, annotations or references for novel families, and updates for the existing ones.
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The basal cell carcinoma
Posted on August 27th, 2010 No commentsBasal cell carcinoma (BCC) is the most diffused tumor among the Caucasian population. Its more frequent in men than in women and usually arises in the fifth decade. BCC involves keratinocytes and keratinocyte stem cells that are present in the epidermis and in the follicular hair. These cells acquire some genetic mutations, generating the transforming phenotype. Based on the current data available in literature, it is not totally understood which genomic alterations are crucial for BCC development. By contrast, multiple genes seem altered in this kind of cancer. Firstly, the hedgehog signaling pathway is considered as the most frequently mutated pathway. In particular ptch1 gene that normally acts as a tumor suppressor is inactivated. Therefore, HH signaling could be a promising target for a pharmacological therapy. Inhibitors are already in clinical trials and seem to have good results and positive perspectives.
Another class of gene that is frequently altered in BCC encodes for the damaged DNA repairing proteins. Of course, as well as in other skin cancer, some mutations in p53 gene have been reported also in the case of the basal cell carcinoma. It is not surprising that BCC could arise in children affected by Xeroderma pigmentosous during the childhood. The polymorphism of the gene of the receptor of the melanocyte stimulating hormone is a risk factor. Indeed, the occurrence of BCC is higher in presence of a certain single nucleotide polymorphism that is responsible for the red or fair phenotype. In fact, in individuals with red or fair hair and pale skin, the pheomelanin is less protective against the UV damage than the eumelanin (the black or brown type) and these persons are more prone to skin cancer, in general. Nevertheless, it has not completely clarified what is the role of UVR damage in the genesis of the basal cell carcinoma.
Data reported in literature are quite contradictory. It seems that there is a correlation between sun exposure and BCC arising, but the systematic use of UV screens doesn’t change the rate or the likelihood to have this kind of cancer. Again, it seems that is the sporadic sun exposure –one or twice a week during jogging or cycling- the most dangerous. Further studies must be done to better clarify this point and give some suggestions to the general population in order to prevent the BCC. Indeed, in terms of economic and social cost the BCC is really expensive and even if normally is not lethal, as the other skin cancer, is aggressive and disfiguring. Surgeon and radiotherapy are the current treatment for the BCC. The great advances of medicine in this field has given satisfactory results either from a clinical point of view and quality of life. Further studies will optimize these techniques and improve the cosmetic outcome. -
The PINEM technology
Posted on August 23rd, 2010 No commentsThe imaging techniques have done important advances and fine cell details have been resolved with high definition. Anyway, the study of certain processes or surfaces is sometimes limited because of the requirement of gold coat or permeabilization treatment that disrupt cells and organelles. At the California Institute of Technology a novel kind of microscopy has been set up. This techniques, namely PINEM (photon induced, near field electron microscopy), uses the combination of photons and electrons beam to resolve structure of nanometer within few femtoseconds. The capture an image only when a photon and an electron beam coincide on the cell or structure or membrane that have to be studied. The first work has been published on protein vesicles in Escherichia Coli cells: the high resolution allows to distinguish the space between the inner and the outer membranes. The polarization of laser and the orientation of cellular layers are two factors that can be regulated in order to achieve image of a single structure. The PINEM technology is being marketed by an electron microscopy company and it belong available to other labs. At the moment the power of resolution is at nanometer level, but the California Institute’s scientists are confident of further improvements.
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The binary expression system Q
Posted on August 19th, 2010 No commentsThe binary expression system is a strategy to finely tune the tragene expression in a model organism. One example of this kind of system is the GAL4- UAS in which the GAL4 gene is under control of the promoter of interest. GAL 4 activates a second exogenous gene bearing the upstream activating sequence. A possible alternative to the GAL 4- UAS system is the Q system. In this case the transcriptional activator QF binds to a specific sequence Q- UAS, as well as its repressor QS. QS in turn can be regulated by the addition of quinic acid into the medium and a further level of control can be added. A common application for this kind of systems is the generation of mosaic animals through the MARCM, the mosaic analysis with a repressible cell marker. In MARCM cell division is coupled to the repression of the gene of interest in one of daughter cells, allowing to study at the same time the effects of knock down in comparison to the normal cells. The Q system works in mammal cells but further improvements are necessary to use this tool for generating transgenic mice or for some application to zebrafish and worm, rather than flies.
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Novel application of live-cell imaging
Posted on August 17th, 2010 No commentsThe reprogramming of somatic cell is a difficult process and the very low rate of success is the challenge that scientists must overcome. Researchers at the Harvard Stem Cell Institute set up a live cell imaging system to follow the differentiation process step by step. They used mouse fibroblast as a model. These cells had been previously transduced with viruses carrying the expression of Oct4, Sox2, c-Myc and Nanog under control of doxycycline. In order to recognize specific lineage during reprogramming, scientists introduced also a doxycycline- inducible fluorescent protein. After induction, they took pictures every 6-12 hours to identify the pluripotent stem cell colonies. They traced many colonies back to a group of cells that quickly proliferated and had peculiar characteristics in terms of size and shape. This observation is in contrast with previous ones because it seems that cells follow an early decision in the ability to reprogramm, rather than a stoichastic event. Nevertheless, this kind of experiment didn’t investigate the presence of a stoichastic event at the beginning of the process, so this hypothesis cannot be ruled out without further studies. Anyway, the live imaging gave important information and other advances in this technique can help to deepen the knowledge of cell dynamics.
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The siRNA principle and some suggestions to design good siRNA
Posted on August 11th, 2010 No commentssiRNA is a gene-regulatory mechanism triggered by dsRNAs. siRNAs which consist of duplexes of 21-23nt RNAs that are base paired with 2-nt 3’ overhangs and is complementary to target mRNA, mimic intermediates of the natural processing of longer double-stranded RNA triggers by RNA-polymerase III. RNA-polymerase III and other component of RNAi machinery specifically recognize an siRNA duplex and selectively incorporate one of the siRNA strands into different RISCs (RNA-induced silencing complex), including the catalytic endonuclease-containing complex, which is responsible for the strong gene-knock down effect. The strand antisense to the targeted mRNA is often referred to as guide strand and its base-paired sense strand is named passenger strand, which is destroyed upon the incorporation of the guide strand into RISC. The catalytic RISC recognizes mRNAs containing perfect or near perfect complementary sequence and cleaves the mRNA at the site precisely 10nt upstream of the nucleotide opposite the 5’-most nucleotide of the guide strand. The mRNA fragments are subsequently degraded by cellular nucleases, resulting in knockdown of the expression of the corresponding gene.
The selection of siRNA against a gene of interest starts with an annotated target mRNA sequence, including its 5’-3’ untranslated regions (UTRs), splice, polymorphic and allelic variants; nevertheless the coding sequence is most commonly targeted. Several algorithms have been developed in recent years that rely on intrinsic sequence and stability features of functional siRNAs; after selection, each candidate siRNA is examined for similarity to all other mRNA transcripts that might unintentionally be targeted at a genome-wide level. It has been demonstrated that structurally symmetric but primary sequence-asymmetric siRNAs from which the target mRNA complementary guide strand has great propensity to be assembled into RISC than the passenger strand, show improved efficacy and specificity. Most of the functional siRNAs have a low to medium G+C content ranging between 30-55%. Indeed, too low G+C content may destabilize siRNA duplexes and reduce the affinity for target mRNA binding, while too high G+C content may avoid RISC loading or cleavage-product release. Additionally, stable duplexes avoid of internal repeats or palindromes are the better silencers.
Single nucleotide positional preferences into siRNA sequence have been identified: U or A at position 1, C or G at position 19, A+U richness between position 1 and 7, these features correlate with the rule of thermodynamic asymmetry and the preferred nucleotides on indicated positions may contribute to the bias for selection of antisense strand; A or U at position 10 may promote catalytic RISC mediated passenger strand and substrate cleavage.
Each strand of the siRNA duplex , once assembled to RISC, can guide recognition of fully or partially complementary target mRNAs, named as on and off targets respectively. Off targets can share contiguous and centrally located sequence complementarity over more than half of the siRNA sequence somewhere within the mRNA sequence, as well as show solely 6 or 7 nucleotides of perfect match preferentially in the 3’UTRs with position 2-7 and 2-8 of the guide siRNA. To enforce the specificity, the current strategy is to select siRNAs in which the strand entering in RISC has some mismatches to all undesired target mRNAs. -
Novel perspectives in diabetes treatment
Posted on August 5th, 2010 No commentsThe “glitazones” family is a group of drug used to treat and care diabetes. The mechanism of action involves the nuclear receptor PPAR-γ: this protein is activated by glitazones, translocated into the nucleus and activates the transcription of genes important for glucose metabolism. Even if the glitazones have great effect in managing diabetes, several severe side effects, such as the increased likelihood to have heart attack, induce the FDA advisory committee to recommend some restriction in glitazones sales or to remove them from market. Scientists from the University of Texas Southwestern demonstrated that the glitazones drug not only activated PPAR-γ, but also inhibited its phosphorylation. This lack of phosphate group seems responsible of side effects. Thus, it could be possible to reduce the side effects by isolating molecules that activates PPAR-γ without interfering the phosphorylation. This preliminary study has immediately gained interest in the scientific community and pharmaceutical companies. Indeed, type II diabetes is largely diffused in the population and it is an expensive problem for the governmental health department that must be quickly solved. The availability of novel drugs and the improvement of old ones, such as metformin and insulin, will offer good therapies to overcome diabetes problem. Of course, the prevention and a correct diet will be useful to prevent this disease.
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La7 cell line as a model to study mammay gland development
Posted on August 2nd, 2010 No commentsLA7 cell line had been isolated from DMBA induced adenocarcinoma in rat several years ago. Up to date, it represents an important model to study the differentiation process of mammary gland. It has been shown that these cells form fluid filled blisters, called domes, by the local detachment of the cellular layer. Dome formation is dependent on a polarized phenotype, the presence of tight junctions and the ability to direct secretion and involves cellular changes similar to those occurring in tubuli and alveoli formation during mammary gland development and in pregnancy. For this reason, La7 seems a good model to study the molecular mechanism of lactation and breast development. Indeed, these cells may be used to identify substances that stimulate milk production. Prolactin and other hormones important for lactation may be added to cells medium and their effects may be quantitavely determined. Once setting up a good test, other molecules may be screened. Lactation is a really important process that naturally occurs. Sometimes, health problems of the mother or child prevent lactation. In these case, an help may be useful to sustain milk production. Of course, this therapeutic intervention must be safe for mother and child and administered only if really necessary.
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The Baculovirus expression system
Posted on July 20th, 2010 No commentsThe Baculovirus expression system provides a versatile and reliable system for the production of recombinant proteins in insect cells. Although many different viruses belong to the Baculovirus family, the most widely used is Autographa Californica multiple capsid nucleopolyhedrovirus (AcMNPV) which can be propagated in Spodoptera Frugiperda (Sf), Trichoplusia Ni (Tn) insect cell lines.Virus genes are expressed in early, late and very late phases of infection: the early and late genes are largely related to the production of viral particles which then bud from the infected cell thus spreading infection to other cells. The very late genes that encode for polyhedrin and p10 are required for the production of the occlusion bodies containing the virus particle in the nucleus of the host cell. Both of these genes are under the control of strong promoter, but can be deleted from the virus genome without interfering with the production of infectious virus particles. As consequence, foreign genes can substitute the polyhedrin and p10 sequences to derive expression vector. This system has been largely used to express mammalian proteins because it guarantees, even if with some limitation, to obtain a correct folding. This capability is extremely important when active enzymes must be synthesized. Further advances in this techniques allow the production of all challenging proteins.
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