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1.
Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy 总被引:8,自引:0,他引:8
The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate
cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical
alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that
a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among
the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes
closest to fulfilling this crucial requirement.
The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a
cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic
adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken
up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken
to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine
during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal
“break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies
of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza
virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape.
Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in
vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive,
easily applicable, widespread technology. 相似文献
2.
K Kuzmov A S Galabov Kh Radeva M Kozhukharova K Milanov 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1985,(6):26-30
The epidemiological effectiveness of dipyridamol, an interferon-inducing agent used for the prevention of influenza and viral acute respiratory diseases, was tested in 4 epidemiological trials, 3 of them carried out as double blind trials. Observations were made in groups of adults (a research institute, a factory) and children (a kindergarten, a school), comprising 1040 subjects in the test groups and 771 subjects in the control groups. The drug was used during the whole epidemic period (January--March 1983) according to the following schedule: 1 oral administration in 8 days, in doses of 8 mg for adults, 50 mg for schoolchildren and 24 mg for children in the kindergarten. The epidemiological effectiveness of the drug was evaluated by comparing the total morbidity rates in influenza and acute respiratory diseases in the test and control groups. The results of 4 trials showed a pronounced epidemiological effectiveness of dipyridamol. The values of the epidemiological effectiveness index of the drug were 2.38 in the kindergarten, 1.55 at the school, 7.42 at the factory and 2.16 at the research institute. The results of the study of dipyridamol suggest that further investigations should be made with a view to use it for the mass prevention of influenza and acute respiratory diseases. 相似文献
3.
A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma
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KeeMing Chia Gavriel Grossman Jonathan J. Ellis Mareike Dahmer‐Heath Dubravka Škalamera Sandra Pavey Bryan Burmeister Brian Gabrielli 《Pigment cell & melanoma research》2016,29(3):329-339
Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. 相似文献
4.
Wouter?van Wyngaardt Teresiah?Malatji Cordelia?Mashau Jeanni?Fehrsen Frances?Jordaan Dubravka?Miltiadou Dion?H?du PlessisEmail author 《BMC biotechnology》2004,4(1):6
Background
Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers.Results
With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA.Conclusion
The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.5.
Skrinjarić I Skrinjarić K Vranić DN Majstorović M Glavina D 《Collegium antropologicum》2003,27(2):753-759
Hypohidrotic ectodermal dysplasia (HED) is characterized by clinical manifestations of severe hypodontia or anodontia, hypotrichosis, hypohidrosis, and specific facial appearance. Affected males show complete expression of clinical features of this condition. Their mothers, who are gene carriers, express only some signs, which are usually very mild. Currently available clinical methods are not sufficient for routine identification of the HED heterozygous gene carriers. The purpose of this study was to identify and describe the facial characteristics of HED patients and their mothers and to evaluate the usefulness of craniofacial pattern profile analysis (CFPP) in the diagnosis of this syndrome and the detection of gene carriers. In this study six affected males and their mothers were evaluated. Z-scores for each variable were calculated and compared with age- and sex-matched controls. Anthropometric analysis showed a specific dysmorphic pattern in CST patients that includes decreased skull base width (t-t: -1.67 Z); decreased forehead width (ft-ft: -1.8 Z), decreased midface depth (sn-t: -2.02 Z), markedly decreased total facial height (n-gn: -3.4 Z), and markedly decreased maxillary arc (t-sn-t: -2.5 Z). Gene carriers showed a similar tendency in their pattern profiles. They showed the same tendency towards lower Z-values for forehead width, facial height, and mouth width. The values for these measurements were between those of the affected and healthy controls. The most pronounced findings were increased head width (eu-eu: +2.83 Z), increased lower face width (go-go: +2.06 Z), and reduction of total facial height (n-gn: -0.95 Z). They also displayed increased nose width (al-al: +2.41 Z) and increased biocular distance (ex-ex: +2.01 Z). When used in conjunction with other methods the anthropometrics pattern profile analysis can considerably enhance detection of gene carriers for HED and increase objective assessment of the craniofacial region in HED patients. 相似文献
6.
The aim of the study was to investigate the range of mandibular movements and to analyze the difference in range of mouth opening, right and left lateral movements, and protrusive movement between patients with clinical diagnoses of temporomandibular disorders and asymptomatic subjects (control group) in a young male population. A total of 240 subjects, aged 19-28, were included in the study. The TMD sample comprised 180 patients (60 patients with muscle disorders; 60 patients with disc displacement with reduction; and 60 patients with muscle disorders and disc displacement with reduction) and was compared with 60 healthy control subjects. All participants were evaluated by the attending dentists at baseline by means of a physical examination of the masticatory system and a history questionnaire which included the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis I measures. Analysis of variance (ANOVA) with the post hoc Bonferroni criteria showed significant difference in ranges of mandibular movements between and within the groups of asymptomatic subjects and TMD patients for active mouth opening (p = 0.001), right lateral movement (p = 0.002), left lateral movement (p = 0.006), and protrusive movement (p = 0.05). It has been found that there are statistically significant differences in the range of mandibular movements that separate asymptomatic subjects and patients with muscle disorders and disc displacements with reduction in this young male population. However, we cannot conclude that measurements of active mandibular movements can discriminate one group (TMD patients) from the other (asymptomatic subjects), because the mean ranges of these active movements between the groups were measured in clinically "normal" values. 相似文献
7.
Genetic diversity and forensic parameters based on 15AmpFlSTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) were evaluated in a sample of 101 unrelated, autochthonous adults from Montenegro. After applying Bonferroni correction, the agreement with Hardy-Weinberg equilibrium (HWE) was confirmed for all loci with the exception of D5S818 (chi2 test) and D21S11 (exact test). The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 studied loci were 0.9999999999999999844 and 0.99999382, respectively. According to measures of within-population genetic diversity, D2S1338, D18S51 and FGA may be considered as the most variable and most informative markers for forensic testing and population genetic analyses out of the 15 analysed loci in a population of Montenegro. D5S818 showed to be the least variable and together with TPOX, the least informative. Interpopulation comparisons were carried out and levels of genetic differentiation between population of Montenegro and five South-eastern European populations (Kosovo Albanians, Serbians from Vojvodina province, Macedonians, Bosnians and Croatians) were evaluated. The most differentiated population in relation to Montenegro is a population of Kosovo Albanians as suggested by both AMOVA and coefficients of genetic differentiation (F(ST) and R(ST)). 相似文献
8.
Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as
the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale.
In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic
Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot
analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate
that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for
the manufacture of therapeutic and other high value proteins. 相似文献
9.
Dubravka Vu?i?evi? Olivia Corradin Evgenia Ntini Peter C Scacheri Ulf Andersson ?rom 《Cell cycle (Georgetown, Tex.)》2015,14(2):253-260
Long non-coding RNAs (ncRNA) have recently been demonstrated to be expressed from a subset of enhancers and to be required for the distant regulation of gene expression. Several approaches to predict enhancers have been developed based on various chromatin marks and occupancy of enhancer-binding proteins. Despite the rapid advances in the field, no consensus how to define tissue specific enhancers yet exists. Here, we identify 2,695 long ncRNAs annotated by ENCODE (corresponding to 28% of all ENCODE annotated long ncRNAs) that overlap tissue-specific enhancers. We use a recently developed algorithm to predict tissue-specific enhancers, PreSTIGE, that is based on the H3K4me1 mark and tissue specific expression of mRNAs. The expression of the long ncRNAs overlapping enhancers is significantly higher when the enhancer is predicted as active in a specific cell line, suggesting a general interdependency of active enhancers and expression of long ncRNAs. This dependency is not identified using previous enhancer prediction algorithms that do not account for expression of their downstream targets. The predicted enhancers that overlap annotated long ncRNAs generally have a lower ratio of H3K4me1 to H3K4me3, suggesting that enhancers expressing long ncRNAs might be associated with specific epigenetic marks. In conclusion, we demonstrate the tissue-specific predictive power of PreSTIGE and provide evidence for thousands of long ncRNAs that are expressed from active tissue-specific enhancers, suggesting a particularly important functional relationship between long ncRNAs and enhancer activity in determining tissue-specific gene expression. 相似文献
10.
PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies. 相似文献