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Ina preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the fragment or variant of. In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment, or variant of albumin, via recombinant DNA technology or genetic engineering. The present invention further includes transgenic organisms modified to contain the nucleic acid molecules of the invention, preferably modified to express the albumin fusion proteins encoded by the nucleic acid molecules. Under these conditions, hGH has no observed Figure 2 depicts the extended shelf-life of an HA fusion protein in terms of the stable biological activity Nb2 cell proliferation of - HA-hGH remaining after incubation in cell culture media for up to 3 weeks at 4, 37, or 50 C. Data is normalized to the biological activity of hGH at time zero. Figure 3A shows proliferation after 24 hours of incubation with various concentrations. Thus in the present application, the drawing is labeled pPPO and more restriction sites of the same vector are shown. Figure 5 compares the recovery of vial-stored HA-IFN solutions of various concentrations with a stock solution after
When stained with ConA, the rapidly replicating tachyzoite forms that cause the acute phase of the disease toxoplas- mosis revealed a fluorescence signal covering the entire body of the tachyzoites with a few dots of cytoplasmic punctate FIG.
Confocal microscopy analysis of lectin-staining formaldehyde-fixed T. Extracellular tachyzoites were purified from infected human foreskin fibroblasts, and bradyzoites were isolated from brains of mice chronically infected by T. Panels 2, propidium iodide staining of nucleus red. Panels 4, merged signal between propidium iodide staining and lectin labels. No specific or obvious labeling of late secretory organelles could be distinctly observed Fig.
The lectin PSA also gave identical cytoplasmic pat- terns in tachyzoites Fig. We also investi- gated the presence of lectin-stained glycoproteins in the dor- mant encysted bradyzoite forms that are responsible for the chronic phase of the disease. The released bradyzoites ob- tained from2-month-old cysts isolated fromchronically infected mice also positively stained with both ConA and PSA, suggest- ing the presence of N-linked glycoproteins in the dormant form of T.
We cannot rule out that N-glycoproteins produced in tachyzoites or at the begin- ning of bradyzoite differentiation in younger bradyzoites are stably conserved throughout the life cycle of encysted brady- zoites. With the exception of lectin signals scattered throughout the whole body of the parasite and surrounding the nucleus Fig.
Most importantly, a strong co-localization with T. In addition, no con- vincing co-localization was observed when polyclonal anti- bodies specific to gliding-associated protein 45 Gap45 panel and myosin light chain MLC panel, two markers of the glid- eosome located between the inner complex membrane and the parasites plasma membrane, were tested. Because it has been described that one major component of the glideosome, TgGAP50 is N-glycosylated 19, the lack of convincing co- localization of lectin fluorescence with glideosome proteins might be explained by the weakness of the lectin signal.
Colocalization of ConA fluorescence with different T. Double fluorescence assays were performed using ConA revealed with FITC-conjugated streptavidin followed by incubation with monoclonal or polyclonal antibodies specific for protein markers of rhoptries Ron1, microneme Mic1, dense granules Gra1, apicoplast Api, actin Act, MyoA, gliding-associated protein Gap45, and myosin light chain 1 MLC. The signals corresponding to monoclonal and polyclonal antibodies were revealed by a secondary goat Alexa Fluor conjugated antibody red.
The images were examined and photographed on a Zeiss confocal microscope. Interestingly an apparent co-localization was observed with the apicoplast, a plastid-like organelle found in apicom- plexan parasites depicted in Fig. As it has been described that proteins translocated into apicoplast are transported across the endoplasmic reticulum 29, we hy- pothesize that some apicoplast proteins containing potential glycosylation sequons may in fact be N-glycosylated.
This issue will be further discussed. Nature of N-linked Glycoproteins Binding to ConAIt was difficult to firmly establish which of the parasites specific organelles and N-glycoproteins were labeled by fluorescence using these lectins. Affinity purification therefore was carried out for the isolation of N-linked proteins that bind to ConA. After five successive washes were tested for efficiency Fig.
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Interestingly only glycoproteins ranging from 45 kDa to higher than 100 kDa were identified Fig. The specificity of N-glycosylated proteins binding to ConA was demonstrated by a competition assay performed by in- cubating detergent protein extracts in the presence of 0. Alternatively the elution was directly performed with the same inhibitor lane 7 instead of SDS sample buffer lane 5.
The disappearance of numer- ous glycoproteins when the competition assay was performed or their appearance during the specific elution with 0. Then large scale preparation was performed using total detergent extract from 10 10 tachyzoites.
Thirteen gel slices bands were excised from the gel as indicated in Fig. As the predicted proteome of T. This approach allowed the identification of 11 additional proteins that were not present in the NCBI protein database of T. In many cases, the same protein was detected in multiple gel slices as mentioned above presum- ably due to carryover of high abundance proteins from one band to another during SDS-PAGE because of partial degra- FIG. A, the pilot experiment performed with detergent extract from 10 9 tachyzoites.
B, large scale purification of ConA-binding glycoproteins using detergent extract from 10 10 tachyzoites. The contaminated proteins corresponding to different ConA species eluted from the beads are indicated by arrows.
MM, molecular mass in kilodaltons. Where a protein was de- tected in multiple gel slices column designated Gel bands and multiple MS data sets were obtained, all the matching peptides identified are shown column designated Se- quences.
Further experi- mental verification of proteomics data using Western blot analyses also validated the presence of different proteins such as myosin A, GAP45, and rhoptry proteins in the T.
maitrise de l`instrumentation, applications et étude des glycosylations
The three surface proteins including the minor glycosylphosphatidylinositol-anchored surface gly- coprotein gp23, which has been described previously as an N-linked glycosylated protein 17, were not detected in the ConA-binding glycoproteins data not shown.
This suggests that either lower amounts of gp23 or its particular N-glycan modifications might explain the absence of gp23 identifica- tion.Needs reliable and noninvasive tools, spheroids could help to (15 mM Tris HCl , pH , M NaCl, 5 mM MgCl2, 10 mM CaCl2, and unsolved disease, meaning that no recovery treatment exists and only the eradication of Atomic Force Microscopy: Application to Investigation of. microscopy image of a representative bacterial film showing a homogeneous and of effective genetic tools for the construction of isogenic mutants in this organism. mM L-glutamic acid (pH ), ml of 1 mM -NAD, ml of deionized water, and ml of. o Micro injection du peptide Pc dans des cellules MCF10A. the cells were allowed to recover for one hour at 37 C in 5 replaced by 10 mM Tris-HCl, pH , M NaCl, Tween such inhibitors would provide more rapid and reversible tools than couverte d un film doré sur une face.
These results also confirm the absence of co-localization between the ConA signal and the major surface antigen SAG1 using a specific monoclonal antibody data not shown. Taken together, these data are consistent with the presence of a limited number of surface proteins, dense granules, and micronemes in the ConA-binding material analyzed by pro- teomics approaches. Nevertheless many novel potentially N- glycosylated proteins in T.
These components play key roles in the particular gliding motility of parasites that allows T. The sec- ond class of N-linked glycosylated proteins that we identified are components of the late apical secretory organelles, named rhoptries Table III. An- other category of secreted proteins present in the rhoptry was also identified Table III and supplemental data.
These are known to be released from the rhoptry during host cell inva- sion and participate in the formation of the vacuole in which the parasites multiply Additional bioinfor- matics searches using our in-house genome database see details under Experimental Procedures identified all poten- tially N-linked glycoproteins, and in addition to the putative partners described above, 11 novel proteins were detected proteins marked by asterisk in Table III and supplemental data.
Some of these novel putative N-glycoproteins are highly conserved in the Apicomplexa, implying that they fulfill parallel roles in the biology of these parasites. For example, we identified the Plasmodium homologue of perforin-like pro- tein 1 Ref. Surprisingly HSP70, HSP90, elongation factor 1a, and eukaryotic translation initiation factor 4A were also de- tected supplemental data. However, HSP70 and HSP90 may be involved in the folding and stabi- lization of genuine N-linked glycoproteins of the glideosome, moving junction, or other subcellular compartments of the parasites.
Therefore, one ion of mass MH. These nucleic acid databases were imported into the local Mascot server, and the segments were translated in all possible six reading frames. The other proteins were identified as described in Table II.
Only the doubly protonated parent ion masses show a mass difference of 162 Da corre- sponding to the mass of a mannose. The glycoprotein Tg- GAP50 represents a genuine pellicle component involved in the parasites motile apparatus.
However, the implication of N-glycosylation, not only in gliding motility but also in the biogenesis of the parasites late secretory organelles, was further investigated in greater detail using both cell biology and pharmacological approaches. Instead the tunicamy- cin-treated tachyzoites displayed a normal replication rate until infected host cells were lysed at 48 h postinfection like the control tachyzoites treated with DMSO alone Fig.
As mentioned, the presence of tunicamycin was not required to FIG. The two additional N-glycosylation sequons are underlined and bold. By comparison, first round tunicamycin-treated tachyzoites that received no further treatment during the second round of new host cell infection still grew more slowly.
Tunicamycin Alters Biogenesis of Late Secretory Organelles and the Inner Membrane ComplexTo determine whether tunicamycin could alter the morphogenesis of different secre- tory organelles and other subcellular compartments of T. Tunicamycin alters the mor- phogenesis of T. Tachyzoites treated with tunicamycin or with DMSO alone for 48 h during the first cycle of infection were released and loaded on new monolayer HFF cells and grown for 24 h without drug.
Inhibitory effect of tunicamycin on T. A, the delayed effect of tunicamycin on T. After 2 days of infection, one-fifth of each well of the first cycle treated or untreated parasites was used to infect new wells under tunicamycin or DMSO treatment. The remaining parasites of this first cycle experiment were recovered, washed with PBS, and frozen. After the second cycle of treatment, one-fifth of each well was used again to infect new monolayer HFF cells for the third cycle experiment.
All parasite pellets were lysed and tested for -galactosidase activity. B, comparison of the growth rate of tunicamycin-treated and untreated tachyzoites. Tachyzoites treated with tunicamycin or with DMSO alone during the first cycle of infection as described were loaded onto new monolayer of HFF cells and grown for 24 h in the absence of drug.
The percent distribution of vacuole size number of parasites per vacuole was counted for 100 vacuoles in three independent experiments, and data are expressed as mean S.
The plasma membrane appeared not to be affected in tunicamy- cin-treated tachyzoites Fig. Neither the con- stitutive secretion of dense granules within the vacuole nor the morphology of micronemes was affected in tunicamycin- treated tachyzoites Fig.
In addition, the fluorescence pattern of rhoptries was not significantly changed after tunicamycin treatment Fig. In contrast, the classical fluorescence pattern of the inner mem- brane complex could not be visualized, suggesting that the morphogenesis of this compartment had been altered Fig. A more diffuse cytoplasmic staining was de- tected with anti-myosin, and the inner membrane complex that is known to be labeled by this myosin marker could not be distinguished Fig.
Some treated tachyzoites also showed a few unknown structures when anti-actin was tested Fig. Collectively these data indicate that tunicamycin induces important alterations in the biogenesis of T. The polyclonal antibodies specific to myosin light chain MLC cross-reacted with a novel T. Effect of tunicamycin on ConA binding and identification of novel N-glycoproteins of T. A, total detergent extract from the same number of tunicamycin-treated or untreated control DMSO only tachyzoites was incubated with ConA beads.
The absence of glycoprotein binding to ConA is indicated by arrows. The nature of this novel N-glycoprotein remains to be determined. As expected, when degly- cosylated the 85-kDa protein was unable to bind ConA lane 4.
The precise nature of this novel 85-kDa glycoprotein re- mains to be determined. In addition, we demonstrated that the rhoptry neck protein RON1 was not capable of binding ConA when tunicamycin-treated lane 4 compared with un- treated lysate lane 3. Because of the lower amount of rhoptry neck protein 2 detected by Western blots combined with our identification of only one peptide isolated by proteomics ap- proaches, it was not surprising that we were unable to vali- date the N-glycosylated status of RON2 using total extract of tunicamycin-treated extracts data not shown.
In contrast, we were able to show that actin Fig. Effect of tunicamycin on T. A, effect of tunicamycin on gliding motility of T. Tachyzoites treated with tunicamycin or with DMSO alone Control for 48 h during the first cycle of infection were released and allowed to glide on serum-coated slides. Trails were visualized by staining with anti-SAG1 antibodies followed by a fluorescent secondary antibody.
B, effect of tunicamycin on host cell entry. The tunicamycin-treated and untreated tachyzoites during the first cycle of infection described above were also used to infect new monolayer HFF cells and grown for 24 h. The data are expressed as mean S. C, schematic drawing illustrating the host cell entry by T.
The current accepted model of host cell entry or invasion proposes contributions from both the glideosome and the moving junction. The glideosome is composed of TgMyoA, actin filaments, and membrane anchor myosin XIV or GAP50 and GAP45, which are located between the plasma membrane and the inner membrane complex, whereas the moving junction contains rhoptry proteins that are secreted during host cell entry.
In light of the observations related in this study, we describe that several components of both glideosome and moving junction are potentially N-glycosylated or contain a number of consensus sites suggesting them to be N-glycosylated.
We believe that this post-translational modification may be required for their proper intracellular transport to the pellicle and for protein-protein interactions.
Only a few tachyzoites treated by tunicamycin showed smaller and aborted trails, whereas the untreated tachyzoites incubated with DMSO only that were analyzed under the same experimental conditions were entirely motile as demonstrated by the prominent trail length for each individual tachyzoite Fig. These data on the inhibition of N-glycosylation of components in- volved in parasites gliding motility are consistent with tuni- camycin treatment affecting the host cell invasion by T.
To establish whether tunicamycin was blocking the attach- ment or invasion of host cells, we monitored host cell reinfec- tion after the first cycle of intracellular parasite treatment. To do this, we examined and quantified the number of parasites attached to or invading host cells. Invasion was scored during a 1-h pulse infection followed by 24 h of intracellular growth.
The cell wall or pellicle of apicomplexan parasites like T. Both actin and myosin A homologues have been local- ized to the space between the plasma membrane and the inner membrane, and this glideosome is a key player in mo- tility and host cell entry by T.
Another essential element represents the moving junction, a structure built at the interface between the host cell and the parasite during its active entry into any kind of mam- malian cell.
The moving junction is a circumferential zone that forms at the apical tip of the parasite, moves backward, and pinches the vacuole from the host cell membrane 34, 36. Several components of the moving junction are secreted from late secretory organelles such as rhoptries RON2 and mi- cronemes AMA1 depicted in Fig. It is clear that the results presented here identified N-linked glycosylation on several key constituents involved in host-parasite interac- tions.
It has been shown that other eukaryotes can transfer structures other than the largest lipid-linked oligosaccharide precursor, Dol- PP-Glc 3 Man 9 GlcNAc 2 , 20. Thus, we postulate that the paucimannosidic N-linked glycan structures found on T. This also indicates that a part of the endoplasmic reticulum and Golgi trimming and maturation pathways that are highly conserved in other eukaryotes is absent in the parasite. However, only structures of major N-glycans were determined when the total detergent glyco- protein extracts were analyzed.
We therefore cannot rule out the presence of other minor modifications and branching monosaccharides that escaped detection in this study.
We identified a number of known proteins that have not been suspected until now to be N-glycosylated. Conversely N-glycosylation is considered as a rare post-translational modification in apicomplexan parasites because most inves- tigations have focused attention on surface and other anti- gens that are important for host cell-parasite interactions and future vaccine development.
Our data confirmed that the key glideosome membrane anchor myosin XIV also named GAP50, known to be involved in both parasite motility and host cell entry, contains N-glycosylated structures. We report for the first time in T. However, it was somewhat unexpected to find that TgMyoA, another component of the glideosome, was also capable of specifi- cally binding to ConA. We have shown that TgMyoA failed to bind to ConA when the parasites were treated with the N-gly- cosylation poison tunicamycin.
Neither the N-terminal signal peptide nor a transmembrane domain is present in TgMyoA. As stated, no obvious domains of TgMyoA presently explain how it might be translocated across the ER membrane to the lumen where the addition of N-glycans is expected to occur co-translationally 10. However, almost nothing is known about ER translocation machinery in T.
In addition, it has been shown that cytoplasmic and nuclear proteins are N-glycosylated, suggesting the existence of post-translational N-glycosylation mechanisms in higher eukaryotes 46, 47. It remains to be determined whether such a non-conventional N-glycosylated biosynthetic pathway op- erates in T. To our knowledge, almost nothing has been reported on N-glycosylation of myosins and compo- nents involved in the motile apparatus in other eukaryotic cells.
Gliding is a unique form of apicomplexan parasite mo- tility that manifests as either circular spirals or a series of helical turns revolving around the long axis of the body of the parasite It has been suggested previously that the soluble protoglideosome and membrane-associated Tg- GAP50 are transported separately and are assembled into the glideosome in the inner membrane complex of mature para- sites 19.Les morpholinos (Gene Tools) sont des oligomères analogues synthétiques M Tris-Hcl pH M NaCl Tween ). The tunicamycins: useful tools for studies on glycoproteins. Catégories du logiciel: Sauvegardes . of Su(H)DBM on Evi1 but is not sufficient to restore the expression. Le film est développé après une nuit à température ambiante. Dernière modification: FRAP: Fluorescence Recovery After Photobleaching. single text file from The Institute for Genomic Research, and seg- mented into B containing 10 mM TrisHCl (pH ), M NaCl, 1 mM CaCl2, 1.
The N-linked glycosylation of glideosome compo- nents such as GAP50 suggests that this post-translational modification may be involved in their intracellular trafficking mechanisms or in their transport to the parasites space where the glideosome are assembled. These moving junction proteins also contain putative N-glycosylation sites.
In addi- tion, we showed that another rhoptry neck protein, RON1, with presently unknown function may be N-glycosylated. It should be mentioned that RON1 displayed the strongest flu- orescence that co-localized with the ConA signal, and its deglycosylation by tunicamycin prevented lectin binding. We were not able to confirm that RON2 and AMA1 are N-glycosylated using West- ern blots because of the limited amount of protein detected after lectin purification.
Further biochemical analyses including purification of higher amounts of RON2 and AMA1 by affinity purification with their respec- tive specific antibodies followed by direct determination of N-glycan structures using mass spectrometry are required. Many other proteins have been isolated by ConA affinity pu- rification and identified by proteomics analyses.
Among these are also proteins that do not have any predicted N-glycosy- lation sites supplemental data, suggesting that these pro- teins may interact with other genuine N-glycosylated proteins.
This may be the case for MIC1 supplemental data that has been reported previously as a parasite lectin 48 that might be retained on ConA by interacting with N-glycan structures carried by other N-glycoproteins that specifically bind to ConA. It is intriguing that unlike all other eukaryotic cells, T. The growth defect appeared only during the second cycle of reinvasion of host cells with a very prominent effect during the third cycle. The effect of tunica- mycin took place in the absence of drug during the second and third cycle of host cell infection, suggesting that this delayed effect, or unusual kinetics of tunicamycin on T.
This conclusion is supported by the results that demonstrate the ability of tunicamycin to affect gliding motility of tachyzoites released after intracellular drug treatment.
We cannot exclude that host cell attachment may also be affected by tunicamycin treatment. However, the lack of N-glycosylation of several SAGs and most MICs reported by lectin purification and pro- teomics analyses indicates that any interference of host cell attachment by tunicamycin may be marginal.
Therefore, we suspect that it is the parasites motility and new host cell invasion that are likely impaired in tunicamycin-treated tachyzoites. It remains to be determined whether moving junction formation is also inhibited by tunicamycin and ab- sence of N-glycosylation.
However, the delayed effect on intracellular replication of tunicamycin-treated parasites that have entered new host cells is highly reminiscent of the de- layed death phenomenon seen with apicoplast inhibitors 49.
It appears that ConA fluorescence co-localizes with the signal of apicoplast-specific monoclonal antibodies.
Because it has been described that some apicoplast proteins transit through the ER 29, 50, it is likely that this plastid-like organelle may also contain N-glycosylated proteins and that the delayed effects seen with tunicamycin might be partially attributable to inhibition of apicoplast functions. In summary, the current study illuminates a diverse reper- toire of N-glycosylated proteins that contribute to parasite survival and pathogenesis during mammalian host cell infec- tion.
Novel parasite-specific N-glycoproteins identified here can significantly expand the number of potential targets for therapeutic intervention. We anticipate that ongoing in-depth exploration of biological functions of the identified N-linked Host-Parasite Interactions Mediated by T. Erreur de chargement Czech.
La plupart des erreurs LNG sont dues à des fichiers manquants ou corrompus. Under these conditions, hGH has no observed Figure 2 depicts the extended shelf-life of an HA fusion protein in terms of the stable biological activity Nb2 cell proliferation of - HA-hGH remaining after incubation in cell culture media for up to 3 weeks at 4, 37, or 50 C. Data is normalized to the biological activity of hGH at time zero.
Figure 3A shows proliferation after 24 hours of incubation with various concentrations. The term "germ cell line transgenic organism" refers to a transgenic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring- the ability- of the traasgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or -all of that alteration or genetic information, then they too are transgenic organisms.
The alteration or genetic information may be foreign to the species of organism to which the recipient belongs, foreign only to the particular- individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
Transgenic -animals can be produced by a variety. The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U. A number of recombinant or transgenic mice have- been produced, including those which express an activated oncogene sequence U.
While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
Animal Science 75 Promoters that control the genes encoding milk proteins are preferred, for example the promoter for casein, beta Iactoglobulin, whey acid protein, or lactalbumin see, e. Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art e.
Methods for generation of genetically engineered plants are further described in US Patent No. Pharmaceutical or Therapeutic Compositions The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral e. The treatment may consist of a single dose or a plurality of doses over a period of time. While it is possible for an albumin fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical-formulation, together with one or more acceptable carriers.
The carrier s must be "acceptable" in the sense of being compatible with the albumin fusion protein and not deleterious to the recipients thereof. Typically, the carriers will be water or saline, which will be sterile and pyrogen free. Albumin fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf- life in solution.
For instance, pharmaceutical compositions of the invention may be formulated well in. For example, wherein the Therapeutic protein is JGH, EPO, alpha-IFN or beta-1 FN, formulations containing the albumin fusion protein may be prepared taking into account the extended shelf-life of the albumin fusion protein in aqueous formulations.
As exhibited in Table 2, most Therapeutic proteins are unstable with short shelf-lives after formulation with an aqueous carrier. As discussed above, the shelf-life of many of these Therapeutic proteins are markedly increased or prolonged after fusion to HA. Powder should be stored Serono im at Rm Temp. After reconstitution store 4 - 8 C for up to 14d.
Specifically, aerosol includes a gas-borne suspension of -droplets of an albumin -fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist-sprayer. Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. For instance,- for human use, both the Therapeutic protein and albumin portions of the albumin fusion protein will typically be human.
In some cases, wherein either component is non human-derived, that component may be humanized by substitution of key amino acids so that specific e-pitopes appear to the human immune system to be human in nature rather than foreign. Such methods include the step of bringing into association the albumin fusion protein with the carrier that constitutes one or more accessory ingredients. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze-dried lyophilised condition requiring only the addition of the, sterile liquid carrier, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders. Dosage formulations may contain the Therapeutic protein portion at a lower molar concentration or lower dosage compared to the non- fused standard formulation for the Therapeutic protein given the extended serum half-life exhibited by many of the albumin fusion proteins of the invention.
Growth hormone is typically administered at 0. As described above, formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.
Albumin fusion proteins of the invention can also be included in nutraceuticals. For instance, certain albumin fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses albumin fusion protein.
Such compositions can also include plant or plant products obtained from a transgenic plant which expresses the albumin fusion protein. The "effective amount" for purposes herein is thus determined by such considerations. More preferably, this dose is. An intravenous bag solution may also be employed. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal. Sustained-release matrices include polylactides U.
Ordinarily, the liposomes are of the small about Angstroms unilamellar type in which -the. Other controlled release systems are discussed in the review by Langer Science For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
The carrier suitably contains minor amounts of additives such as substances that. The albumin fusion protein is typically formulated in such vehicles at a concentration. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts. Sterility is readily accomplished by filtration through sterile filtration membranes e. In a specific and preferred embodiment, the Albumin fusion protein formulations comprises 0.
In another specific and preferred embodiment, the Albumin fusion protein formulations consists 0. Finally, polysorbate has been added as a generic surfactant, which lowers the surface tension of the solution and lowers non- specific adsorption of the albumin fusion protein to the container closure system. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.
Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.Brazilian compare.xsl File Recover PC Tools, Téléchargement recommandé (WinThruster): Optimisez votre PC et réparez LNG Windows C: Program Files (x86) File Recover ugLng . medium was removed and ml of extraction buffer ((20 mM HEPES pH , M NaC1, 1 Triton X , SDS, 2 mM Na3VO4, 2 niM Na4P2O7 and a. Liste de tous les fichiers avec XSL file extension (Données).
Administration "in combination" further includes the separate administration of one of the- compounds or agents given first, followed by the second. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in 93 CA combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin.
In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, -plasminogen, lys-plasminogen, -alpha2-antiplasmin, streptokinae e.
In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole e. Additional protease inhibitors. Additional antiretroviral agents include integrase inhibitors. Other immunosuppressive agents that. In a-specific embodiment, immunosuppressants may be used to.
In an additional embodiment, the compositions of the invention are administered alone 25 or in combination with an anti-angiogenic agent. Such transition metal species may form transition metal complexes. Representative examples -of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten- and molybdenum complexes also include oxo complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten IV oxide and tungsten VI oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates.
Suitable molybdenum oxides include molybdenum VI oxide, molybdenum VI oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars. A wide variety of other anti-angiogenic factors may also be utilized within the context of the.
Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell- extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells.
Other anti- angiogenic agents act to indirectly inhibit angiogenesis. In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein.Amedee. Belafsky, P.C. Fitzpatrick, P.C. "Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification "Quantitative Analysis of Proteome Coverage and Recovery Rates for downloaded as a single text file from The Institute for Genomic Research, and Tris HCl (pH ), M NaCl, 1 mM CaCl2, 1 MnCl2 at room temperature. Woodworth, B.A. Godin, D.A. New chemical and biological tools for targeting in cancer imaging and of heparanase solution ( ng mL heparanase in Tris-HCl pH , M NaCl and CHAPS). la mme sans toi, et nos ordi non plus d ailleurs), la tout aussi indispensable Danile Thierse, Fabrice Bertile (les amricains pendant ce temps l), Cyril Colas. and recovery from ischemic stroke. Scandurro, A.B.
In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic - -agent. Chemotherapeutic agents that may.
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab also known as RemicadeTM Centocor, Inc. Ina specific embodiment, compositions of the invention are administered in combination with Rituximab.
In a specific embodiment, compositions of the invention are administered in combination with tositumomab. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs. Zevalint may be associated with one or more radisotopes. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens e. Treatments for gastrointestinal disorders that.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising albumin fusion proteins of the invention. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing. Additionally, molecules encoded within the viral vector, e. These vectors provide efficient delivery of nucleic acids into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
The development of specialized cell lines termed "packaging cells" which produce only 10- replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes for a review see Miller, A. A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.
Protocols for producing recombinant retroviruses and for infecting 15 cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. Greene Publishing Associates, , Sections 9. Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors.
The genome of an adenovirus can be manipulated such that it 20 encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle.
See, for example, Berkner et al. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d or other strains of adenovirus e. Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.
Additionally, introduced adenoviral 30 DNA and foreign DNA contained therein is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional- mutagenesis in situations where introduced DNA becomes integrated into the host genome e.
Moreover, the carrying capacity of the adenoviral genorne for foreign DNA is large up to 8 kilobases relative to other gene delivery vectors Berkner et al. Gene delivery systems for a gene encoding an albumin fusion protein of the invention can be introduced into a patient by any of a number of methods.
For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter see U.
Patent 5,328,470 or by Stereotactic injection e. The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Additional Gene Therapy Methods Also encompassed by the invention are - gene therapy methods for treating or preventing disorders, diseases and conditions.
This method requires a polynucleotide which codes for an albumin fusion protein of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the fusion protein by the target tissue. Thus, for example, cells from a patient may be engineered with. Such methods are well-known in the art. For example, see Belldegrun, A. Cancer 114 CA Inst. In one embodiment, the cells which are engineered are arterial cells.Dernière modification: Télécharger la dernière version de File Recover version pour Besoin d aide sur ce logiciel File Recover Créez un USB exécutable pour DOS. Catégories du logiciel: Sauvegardes .
The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection. As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues heart, muscle, skin, lung, liver, and the like. The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In one -embodiment, polynucleotides encoding the albumin fusion proteins of the present invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. Such methods are described, for example, in U.
The polynucleotide vector constructs used in the gene therapy method are. Other suitable vectors will be readily apparent to the skilled artisan. Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. The promoter also may be the native 115 CA promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen - fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone.
It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells.
They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such. For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
However, -other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous -membranes- of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure. CA known in the art. The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc.
Such methods of delivery are known in the art. In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic positively charged, anionic negatively charged and neutral preparations.
However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
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Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA Feigner et al. Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. Similar methods can be used to prepare liposomes from other cationic lipid materials. How to install operating system without bis on blackberry For those seeking a pleasant theme for their display, this creation has you covered.
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