Low Molecular Weight DNA Research Articles

The value of blood plasma low-molecular-weight DNA for diagnostics of pathological processes of different genesis

The low-molecular-weight DNA appears in blood plasma a few hours after exposure of rats to ionizing radiation, and its content correlates directly with the irradiation dose. Cloning has shown, that the lowmolecular-weight DNA is enriched with C + G pairs and features of its primary structure characterize its capacity to form rather stable nucleosomes. DNA isolated after irradiation of rats with principally different doses 8 and 100 Gy differed not only quantitatively, but also by the content of the dinucleotides CpG and CpT; this suggests that they originate from different regions of the genome. It has been shown for the first time that exposure of animals to low-frequency noise results in an increase of the content of blood plasma low-molecular-weight DNA. Strokes are characterized by the increase of this DNA during 3 days after the beginning of the acute period, and dynamics of its excretion differs in patients with ischemic and hemorrhagic forms of this disease. In the case of ischemia low-molecular-weight DNA appears in cerebrospinal fluid. In contrast to patients with non-obstructive chronic bronchitis, patients with the chronic obstructive pulmonary disease in the state of remission are characterized by decreased levels of blood plasma low-molecular-weight DNA. The clear dependence between formation and specific features of the low-molecular-weight DNA fraction in blood plasma suggests that it may serve as a universal quantitative marker of apoptosis, which differentiates basically different conditions of the body.

Characterization of the Human SNM1A and SNM1B/Apollo DNA Repair Exonucleases

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Abstract 715: Detection of a p53 codon 249 hotspot mutation in the urine of the patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC), the 5th most frequent cancer worldwide, has a 5-year survival rate of 14% because it is difficult to diagnose early. The goal of this project is to construct a panel of circulation-derived DNA markers to use in a urine test for the early detection of HCC to improve its prognosis. The HCC-specific p53 codon 249T mutation was the first candidate DNA marker used to explore the criteria needed to detect HCC-derived DNA markers in urine of patients with HCC in a sensitive, noninvasive manner. We showed previously that urine contains circulation-derived DNA fragments that are mostly fewer than 300 bp, designated as low-molecular-weight (LMW) urine DNA. The LMW urine DNA contains DNA from tumor tissues when tumors are present. The tumor-derived DNA fragments offer the potential to develop absolutely noninvasive urine tests for the detection of any cancer with known DNA biomarkers. A locked nucleic acid clamp-mediated PCR assay for the p53 249T mutation, targeting only 41 nucleotides of the template, followed by melting curve analysis, was developed and tested using patient urine samples. Total urine DNA samples from 17 patients with HCC were fractionated into high-molecular-weight (HMW) (>1 kb, mostly cell-associated) DNA from the urinary tract and LMW (< 1 kb, mostly circulation derived) DNA and subjected to the p53 249T mutation assay. Encouragingly, samples from 9 of 17 patients with HCC contained detectable p53 249T mutation in the LMW urine DNA fraction and only 1 of 17 matching HMW urine DNA fraction samples was found to contain p53 249T mutated DNA. These findings suggest that the assay target template size of 41bp is suitable to detect HCC-derived DNA markers in urine and that the p53 249T mutation detected in LMW urine DNA is derived from the circulation. Next, we tested a larger number of samples of LMW urine DNA from patients with HCC. Overall, we detected the p53 249T mutation in 50.8% of the LMW urine DNA samples from patients with HCC (60/118). Of the 49 subjects whose urine was positive for the p53 mutation before surgery, the p53 mutation was no longer detectable in the urine of 29 of those patients after surgical removal of the tumor. This result suggested that the p53 249T mutations detected in the urine before surgery were derived from the surgically removed HCC. For controls, we tested LMW urine DNA from patients with hepatitis and cirrhosis and from normal subjects or patients with colorectal cancer and found that 0 of 32 normal (0%), 0 of 31 colorectal cancer (0%), 7 of 69 hepatitis (10.1%), and 6 of 47cirrhosis (12.8%) samples contained detectable amounts of p53 249T mutated DNA. A urine test that includes multiple DNA markers for HCC screening and recurrence monitoring is currently being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 715. doi:1538-7445.AM2012-715

Evaluation of damage to DNA induced by UV-C radiation and chemical agents using electrochemical biosensor based on low molecular weight DNA and screen-printed carbon electrode

There is great interest and need to detect and evaluate damage to DNA by environmental factors. In the present paper, simple electrochemical DNA biosensors composed of commercially available screen-printed carbon electrode (SPCE) and low molecular weight double-stranded DNA (dsDNA) recognition layer are reported and applied to the detection of damage to DNA by UV-C radiation and reactive oxygen species produced by the Fenton type reaction in model as well as mineral water samples with additives. Complex DNA biosensor response is based on square-wave voltammetric intrinsic signal of the guanine moiety as well as that of the intercalative indicator thioridazine, cyclic voltammetric response of the [Fe(CN)6]3−/4− indicator in solution and on electrochemical impedance spectroscopy when the measurements can be performed in the same solution. For the last two types of measurements, the biosensor was also used with an interface between the SPCE and DNA formed by a composite of carboxylated single-walled carbon nanotubes and chitosan to enhance the transducer conductivity. Individual electrochemical/electrical signals depend on the time of biosensor incubation in a cleavage medium and their profiles characterize process of deep DNA degradation.

Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals

The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb).

Blood and Tissue Leukocyte Apoptosis inTrypanosoma bruceiInfected Rats

Severity of disease in human African try- panosomiasis may be linked, as in other human protozoan infections, to apoptosis of host inflammatory cells during infection. The present study investigates the involvement of leukocyte apoptosis in Trypanosoma brucei infection of rodents. Twenty-seven Wistar rats were infected with 1 × 10 3 T. brucei and leukocyte apoptosis was evaluated by several methods. Apoptotic cell count was performed in blood, spleen, thymus, lymph node and liver during infections, using Light Microscopy (LM), agarose gel electrophoresis and Transmission Electron Microscopy (TEM). Blood and tissues leukocyte apoptosis in infected animals were confirmed by LM, TEM, and agarose gel electrophoresis, which showed low molecular weight DNA fragments. Infected rats sacrificed after day 8 PI showed sig- nificant increase in apoptotic cells in both blood ( p< 0.001) and spleen ( p< 0.05). Peak blood leukocyte apoptosis corresponded with peak parasitemia and the lowest total leukocyte count in all infected rats. Our data provide the first documentation of increased levels of blood leukocyte apoptosis during T. brucei infection. Apoptosis of blood cells during trypanosome infection may represent a putative mechanism for the severity of leukopenia and disease.

Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

Abstract 3175: Detection of hypermethylated vimentin gene in urine of colorectal cancer patients

Abstract Although the success of cancer treatment often depends on the early detection of cancer, colorectal cancer (CRC) screening is hindered by low compliance rates (40%) of inconvenient, invasive screening tests. Previous studies have demonstrated that urine contains circulation-derived low-molecular-weight (LMW) DNA (&amp;lt;300 bp) and suggested that LMW urine DNA could be used to detect CRC associated genetic alterations. Aberrant hypermethylation of the vimentin gene (mVIM) has been shown to be a potential stool DNA marker for CRC screening. It was of interest to see whether mVIM could be detected in urine of patients with CRC. In this study, we developed a quantitative MethyLight assay targeting a 39 bp DNA template for mVIM. A locked nucleic acid was incorporated at the CpG sites of the reverse primer of the assay to increase the assay sensitivity and specificity for methylated templates. A blinded concordance study between urine and matched CRC tissue was performed. Twenty matched sets of urine and CRC tissue were collected and subjected to DNA isolation. Total urine DNA was further fractionated into both LMW DNA (&amp;lt; 1 kb) and high-molecular-weight (HMW) DNA (&amp;gt;1 kb) fractions. The MethyLight assay was used to quantify the amount of mVIM in each DNA sample. mVIM was detected in 75% (15/20) of the LMW urine DNA fraction, 0% (0/20) of the HMW urine DNA fraction and 85% (17/20) of the CRC tissue DNA. There was a 60% concordance in detecting mVIM between the CRC tissues and their corresponding urine samples. As negative controls, twenty LMW urine DNA fractions from subjects with no known neoplasia were also tested for mVIM and only 10% (2/20) of control samples contained detectable mVIM. mVIM may be a useful part of a urine based genetic biomarker panel for the early detection of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2011-3175

The Voltammetric Responses of High and Low Molecular Weight DNA on a Variety of Carbon Substrates; Demonstrating the Benefits of Graphitic Surfaces

AbstractThis work compares the voltammetric response of high (∼2000 bp) and low (≤300 bp) molecular weight DNA on a variety of carbon based electrodes demonstrating for both species the advantages of using graphitic electrodes. Results suggest that the edge plane defects are kinetically dominant for the electro‐oxidation of nucleobases, whereas the basal plane sites are favoured for adsorption of the DNA to the electrode surface. For the high molecular weight DNA it is shown that although larger currents are obtained through the use of the edge plane electrode, it is beneficial in terms of signal‐to‐noise ratio to use a basal plane electrode.

“Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H2O2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

Assessing metal, protein, and DNA profiles in Labeo rohita from the Indus River in Mianwali, Pakistan

This study assessed metals in water and different tissues of Labeo rohita and the impact of these metals on DNA and proteins as biomarkers of gills and muscles of these fish from three different polluted sites (reference or low = KW, medium = CH and high = SK) of the Indus River, Pakistan. The Mn, Pb, Cu, Zn, Hg, and Cr levels in water, gills, liver, muscles, and skin of these fish were compared with the international permissible levels. All metals except Pb and Hg in water were within the acceptable limits of drinking water. In contrast, the Mn, Hg, and Cr levels in the fish tissues were higher than their permissible limits for fish as a human food. Here, the gills contained higher metals than the other tissues. Different patterns of biomarkers were found in fish from these sites. While the gills did not show four protein bands (55, 30, 18.4, and 16.4 kDa), the muscles showed four new protein bands (100, 85, 45, and 20 kDa) for fish from the medium and high polluted sites as compared to the reference or low polluted site. The fish from the CH and SK sites of the Indus River contained low molecular weight DNA in their gills but high molecular weight DNA in their muscles when compared with the KW site. This study suggests that the proteins and DNA profiles of L. rohita could be used as biomarkers to assess the impact of potential environmental stressors such as metals on the freshwater systems.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.

DNA fragmentation rate in human thyroid tissue and carcinomas and its change under the influence of a-tocopherol and iodine ions in vitro

The rate of DNA fragmentation was studied in intact thyroid tissue and thyroid carcinoma tissue. The determination of low molecular-weight DNA concentrations and the electrophoretic analysis of DNA isolated from the thyroid tissues and tumors, followed by the calculation of the content of oligonucleosome fractions, indicated a drastic reduction in the DNA fragmentation rate in papillary carcinomas and no its significant changes in follicular carcinomas. In the intact thyroid tissue, α-tocopherol and potassium iodide at a concentration of 10-7 M inhibited the rate of stimulated DNA fragmentation in vitro. The agents showed no effect in the papillary carcinoma tissue and their effect was similar in the follicular carcinoma tissue, but it was less pronounced in intact thyroid tissue.

Leishmania mexicana: Inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells

Macrophages (Mϕ) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect Mϕ and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at ×8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher ( P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher ( P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Immobilizing DNA on clay mineral modified carbon paste electrodes

Al-kaolinite, Al-montmorillonite, and Al 13-montmorillonite have been prepared and their electrokinetic mobility were measured to be – 2.06 × 10 - 8 , -0.62 × 10 - 8 and +2.98 × 10 - 8 m 2V - 1s - 1 respectively. DNA immobilized on the clay mineral modified carbon paste electrode via the positive charges of the clay mineral. The immobilizing efficiencies of DNA were monitored with the DNA markers methylene blue (MB) and ethidium bromide (EB) and they were of the following sequence: Al-kaolinite < Al-montmorillonite < Al 13-montmorillonite. The immobilization did not affect the binding event between DNA and MB or EB. The obtained DNA-sensors can be used to study the interaction between low molecular weight compounds and DNA as well as the different binding strength of low molecular weight compounds with ssDNA and dsDNA.

Insertion of the 1.2kb Chicken β-Globin 5′HS4 Insulator Element in the LTR Inhibits HIV-1 Vector Mediated Transduction at the Early Infection Step.

Insertional oncogenesis is the major concern in gene therapy using integrating vectors such as gamma-retrovirus vectors and lentivirus vectors. One promising strategy to circumvent this unwanted event is the use of insulator elements which can block trans-activation of proto-oncogenes by enhancers of the integrated vectors. The 1.2kb fragment containing the chicken β-globin 5′ hypersensitivity site 4 (5′HS4) is the most well characterized insulator element. The insulator elements are usually inserted into the U3 region of the 3′ LTR of the vector construct, because the insulator element is duplicated and transferred to the 5′ LTR during viral replication. In the transduced cells, two insulator elements are located outside the retroviral transcriptional unit and this configuration is thought to be essential for efficient enhancer blocking. However, we have previously shown that insertion of the 1.2kb 5′HS4 element in the LTR of SIN-HIV-1 vector significantly reduced the transduction efficiency assayed by GFP expression in HeLa cells. In the present experiments, we examined the mechanism of reduced biological titer. We compared SIN-HIV-1 vectors with (INS) or without (PARENT) the 1.2 kb insulator element. The biological titer on HeLa cells (TU/ml), p24Ag concentration (ngp24/ml), and virus RNA genome content (vg copy/ngp24) of these vectors were shown in Table 1. As we previously reported, the biological titer of INS was significantly lower than that of PARENT, although p24 and RNA contents were not influenced by insertion of the 1.2 kb insulator element. In the next experiment, we performed reverse-transcription kinetic assay. Genomic DNA and low molecular weight DNA extracted by Hirt’s method were prepared from transduced HeLa cells and the amounts of reverse-transcribed vector sequence were determined by qPCR. The data showed that reverse-transcription of INS was remarkably reduced compared to PARENT. These results suggest that insertion of the 1.2 kb insulator does not inhibit production of virus particles and packaging of RNA genome. The responsible step for reduced titer of INS appears to be somewhere from the cellular entry step to the reverse-transcription step. Characterization of HIV-1 vector with the 1.2 kb 5′HS4 insulator x106 TU /ml ngp24 /ml x103 vg copy/ngp24 Values are mean ± sem from quadruplicate. a: p&lt;0.005 PARENT 27 ± 4.3 361 ± 56 860 ± 16 INS 4.8 ± 0.4 a 348 ± 37 650 ± 81

Preparations, characterizations and applications of chitosan-based nanoparticles

Chitosan is a natural polysaccharide prepared by the N-deacetylation of chitin. In this paper we have reviewed the methods of preparation of chitosan-based nanoparticles and their pharmaceutical applications. There are five methods of their preparations: emulsion cross-linking, emulsion-droplet coalescence, ionic gelation, reverse micellar method and chemically modified chitosan method. Chitosan nanoparticles are used as carriers for low molecular weight drug, vaccines and DNA. Releasing characteristics, biodistribution and applications are also summarized.