Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 631 records · Page 35Linked to original sources

PubMed1999

Alternative splicing, gene localization, and binding of SH2-B to the insulin receptor kinase domain.

The SH2-B protein is an SH2-domain-containing molecule that interacts with a number of phosphorylated kinase and receptor molecules including the insulin receptor. Two isoforms of the SH2-B have been identified and have been proposed to arise through alternate splicing. Here we have identified a third isoform of the SH2-B protein, SH2-Bgamma, that interacts specifically with the insulin receptor. This interaction required phosphorylation of residue Y1146 in the triple tyrosine motif within the activation loop of the IR kinase and is one of only two signaling molecules shown to interact directly with this residue of the insulin receptor kinase domain. The intron/exon structure of the SH2-B gene was determined. Alternate splice sites utilized to generate the different isoforms of the SH2-B protein were identified in the 3' end of the SH2-B gene immediately downstream of the exon encoding the core of the SH2 domain. Additionally, the chromosomal location of the SH2-B gene was determined to be the distal arm of mouse Chromosome (Chr) 7 in a region linked to obesity in mice.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation. We describe the preparation, purification and characterization of 43 oxygenated sterols, including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F7) and 26,26,26,27,27,27-hexadeuterio (d6) substitution. The 7 alpha-hydroxy, 7 beta-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3 beta, 26-diol (1d) and their 16,16-dideuterio analogs were also prepared. These d2-26-hydroxysterols and [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol (1e) were synthesized from [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol diacetate (2e), which can be prepared from diosgenin. The highly specific deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholesterol samples by isotope dilution methods. The delta 5-3 beta, 7 alpha, 26- and delta 5-3 beta, 7 beta, 26-triols were regioselectively oxidized/isomerized to the corresponding delta 4-3-ketosteroids with cholesterol oxidase. Also described are 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, its 5 beta,6 beta-isomer, cholestane-3 beta, 5 alpha,6 beta-triol, their F7 and d6 derivatives, and d3-25-hydroxycholesterol, which was prepared from 3 beta-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods, including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assignments are presented, and 1H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side-chain protons of 30.

Cholesterol↗

PubMed1999

Expression and characterisation of a Plasmodium falciparum protein containing domains homologous to sarcalumenin and a tyrosine kinase substrate, eps15.

We have identified in Plasmodium falciparum a novel gene encoding a putative bi-functional protein, termed PfPast-1, from genomic and cDNA libraries. Analysis indicated that the sequence encodes a 62 kDa protein of 529 amino acid residues with two distinctive domains: a sarcalumenin-like domain of approximately 320 amino acids at the amino half of the molecule, which shares homology to a major sarcoplasmic reticulum lumenal protein, sarcalumenin, and an eps15 homology domain of about 90 amino acids located at the carboxyl terminus. The eps15 homology domain, first identified in a tyrosine kinase substrate, eps15, and found in increasing numbers of mammalian proteins, has recently been suggested as a protein-protein interaction domain involved in intracellular sorting. Genomic sequences encoding similar proteins containing both the sarcalumenin-like and eps15 homology domains have been identified in humans and Drosophila. RNA blot analysis revealed the presence of a single messenger RNA transcript approximately 3.7 kb in size, which is expressed in all the developmental stages examined with the highest level in extracellular gametes followed by erythrocytic asexual stages, and the lowest in the gametocytes. In the attempt to define its biological function, we have expressed a full-length recombinant PfPast-1 protein in Escherichia coli. Specific immune serum directed against the recombinant protein recognised a approximately 55 kDa protein in the parasite lysate. Further characterisation of PfPast-1 may help in elucidation of its functions in P. falciparum.

Amino Acid Sequence↗

PubMed1999

A new approach to immunological sexing of sperm.

A non-invasive, immunological method for sexing mammalian sperm would be of benefit to agricultural industries. This paper presents a new approach, based on the hypothesis that sex-specific proteins (SSPs) are evolutionarily more highly conserved than non-SSPs. Antibodies to non-SSPs were raised and used in an affinity procedure to remove non-SSPs and enrich for SSPs. Thereafter, using column chromatography, purified SSPs were obtained. Sex-specific antibodies (SSAbs) raised against these SSPs appear to bind to sex-chromosome-specific proteins (SCSPs) on the sperm membrane and make possible a sperm-sexing procedure. Antibodies to SCSPs were raised and used to identify putative SCSPs by affinity chromatography. The preliminary results presented here suggest that a viable immunological sperm sexing procedure can be developed.

Animals↗

PubMed1999

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.

Acid Phosphatase↗

PubMed1999

Further investigations on the hydrolytic degradation of poly (DL-lactide).

The hydrolytic degradation of poly(DL-lactide) (PLA50) material was investigated in order to elucidate the effects of temperature and acidity of the external medium on the degradation characteristics. It was observed that at 60 degrees C and at pH = 7.4, degradation was extremely rapid as compared with degradation at 37 degrees C. After only 2 days, heterogeneous degradation was observed due to larger internal autocatalysis. On the other hand, in the case of degradation at 37 degrees C in an acidic medium with pH = 3.7, the heterogeneous degradation was preceded by a much longer lag time. Water absorption was found to be pH dependent. At pH = 7.4, water absorption was increased due to the osmotic pressure driving the buffer solution into the polymer matrix to neutralize acidic endgroups, which was not the case for degradation at pH = 3.7. In both cases, the oligomeric stereocomplex was obtained as degradation residue at the end of the degradation period.

Biocompatible Materials↗

PubMed1999

A survey of blindness and cataract surgery in Doumen County, China.

PURPOSE: To assess blindness prevalence and that caused specifically by cataract in rural southern China. DESIGN: Population-based, cross-sectional study. PARTICIPANTS: A total of 5342 persons older than 50 years of age. METHODS: Visual acuity and eye examinations were performed in the summer of 1997 in a random sample of villages in Doumen County. Differences in blindness prevalence associated with age, gender, and education were explored using logistic regression. The survey was preceded by a pilot study in which operational methods were refined and quality assurance measures were performed. MAIN OUTCOME MEASURES: Distance visual acuity and lens status. RESULTS: Bilateral blindness (presenting visual acuity < 0.10) was found in 4.37% (95% confidence interval, 3.67%-5.06%). Blindness was associated with increasing age (P < 0.001) and with the lack of education (P < 0.01). Cataract was the principal cause of blindness in at least one eye in 61.5% of blind people, with refractive error responsible for another 10%. An estimated 40% of the cataract blind were operated on; surgical coverage was lowest among the elderly, women, and those without schooling, although not at statistically significant levels. CONCLUSIONS: Despite the increased attention given to eye care in Doumen County, blindness remains a major public health problem. Cataract surgery is reaching fewer than half of those who could benefit from it.

Aged↗

PubMed1999

Visual acuity and quality of life in patients with cataract in Doumen County, China.

PURPOSE: To evaluate the effectiveness of cataract surgery in achieving sight restoration and vision-related quality-of-life (QOL) in patients from rural southern China. DESIGN: Population-based, cross-sectional study. PARTICIPANTS: A total of 109 cataract operated persons (152 eyes) and 654 unoperated persons. METHODS: Cluster sampling was used in identifying a random sample of 5342 persons 50 years of age and older for visual acuity and eye examinations. Visual functioning (VF) and QOL questionnaires were administered to aphakic and pseudophakic individuals, unoperated persons with presenting visual acuity less than 0.10 in either eye, and a sample of those with normal vision. MAIN OUTCOME MEASURES: Distance visual acuity, VF, and QOL questionnaire scores. RESULTS: Among the cataract operated participants, 43 (39.4%) were bilaterally operated on, 32.1% had presenting visual acuity less than 0.10 in both eyes with 8.3% greater than or equal to 0.32 in both eyes. Of operated eyes, 52.6% presented with visual acuity less than 0.10, 23.7% greater than or equal to 0.32; with best correction, the corresponding percentages were 21.1% and 42.1%. Uncorrectable aphakia due to surgical complications was common. In a multivariate regression model, better visual acuity outcomes were associated with higher level surgeon practice settings and recent surgery. On a 0 to 100 scale, mean VF and QOL scores for the cataract operated population were 41.6 and 54.5, respectively. Mean scores ranged from 84.4 and 93.4, respectively, for the unoperated persons with normal vision, to 14.6 and 31.2, respectively, for those with visual acuity less than 0.05 in both eyes. The VF and QOL scores were closely correlated with presenting visual acuity in both cataract operated and unoperated populations (r = 0.49-0.64). Scores among the cataract operated population were not influenced by age, gender, or education level. Among the unoperated population, lack of education was associated with lower VF and QOL scores (P = 0.017 and P = 0.005, respectively), and older age was associated with lower QOL scores (P < 0.001). CONCLUSION: Patients in rural southern China are not realizing the full sight-restoring potential of modern-day cataract surgery. Remedial efforts are needed to improve the performance of local eye surgeons.

Aged↗

PubMed1999

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans.

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.

Analysis of Variance↗

PubMed1999

Pattern of localization of primitive hematopoietic cells in vivo using a novel mouse model.

Bone marrow transplantation is increasingly used as a treatment for numerous immunologic, hematologic, and malignant disorders. However, the mechanism by which transplanted hematopoietic stem cells are engrafted is not completely understood. Many traditional techniques have been used to study the engraftment of transplanted stem cells. Most of these methods are ex vivo and, in some cases, donor cells must be modified to enable detection. We describe a novel alternative for identifying unmodified primitive donor cells in a murine host. This mouse model is based on the differential capacity of adenine phosphoribosyltransferase (APRT)-positive and APRT-negative cells to sequester and incorporate radiolabeled adenine. Aprt is the gene encoding the adenine phosphoribosyltransferase purine salvage enzyme and has been ablated in 129sv mice. Following the injection of APRT-positive c-kit-positive enriched hematopoietic cells into syngeneic, sublethally irradiated APRT-deficient mice, engrafted cells and their presumptive progeny were successfully tracked by polymerase chain reaction. Their presence also was visualized by autoradiography of paraffin-embedded tissue sections. APRT-positive c-kit-positive enriched cells were detected in the bone marrow, spleen, lung, and thymus of nonirradiated mice. Donor cells and their progeny were more widely distributed in tissues of sublethally irradiated mice than of their nonirradiated counterparts, demonstrating that the pattern of localization of c-kit-positive enriched cells differs between nonirradiated and sublethally irradiated syngeneic recipients. The Aprt mouse model provides a sensitive method for further studying the mechanism of engraftment of unmodified donor hematopoietic cells in relation to the tissue architecture of the recipient.

Adenine↗

PubMed1999

Real-time visualization of myocardial perfusion and wall thickening in human beings with intravenous ultrasonographic contrast and accelerated intermittent harmonic imaging.

Previous work has demonstrated that at higher peak negative pressures, microbubbles are destroyed by diagnostic ultrasonography. At lower pressures (lower mechanical index), less destruction occurs but enhanced contrast persists. In animals, this lower mechanical index has resulted in enhanced contrast after administration of intravenous microbubbles with intermittent imaging at faster frame rates. We tested whether this accelerated intermittent imaging technique could produce myocardial contrast and detect myocardial perfusion abnormalities in 25 patients (10 with normal wall motion, 15 after myocardial infarction). Three independent reviewers detected persistent myocardial contrast defects within the infarct zone throughout the cardiac cycle in 9 of the 15 patients after acute myocardial infarction; the presence of such defects was predictive of a persistent regional wall motion abnormality at 4-week follow-up. Interobserver agreement on regional contrast enhancement ranged from 88% to 90%. We conclude that accelerated intermittent imaging permits real-time visualization of myocardial blood flow and wall thickening.

Contrast Media↗

PubMed1999

Effect of transducer standoff on the detection, spatial extent, and quantification of myocardial contrast defects caused by coronary stenoses.

Intermittent harmonic imaging during a continuous infusion of microbubbles may be able to quantify myocardial perfusion abnormalities. Measurements of the spatial extent of these perfusion abnormalities depends on homogenous destruction of the microbubbles in the elevation plane of the transducer. We hypothesized that uneven microbubble destruction caused by attenuation of beam intensity could alter quantitative measurements of perfusion abnormalities during stress. To test this hypothesis, we measured the spatial extent of perfusion defects at peak dobutamine stress with a continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin and intermittent harmonic imaging in dogs with nonflow-limiting coronary stenoses in the left anterior descending artery. The spatial extent of perfusion defects was also measured during total occlusion of the artery. Measurements were made at standoffs of 2- to 3-cm and 4- to 5-cm distance from transducer surface to myocardium. These spatial extents were correlated with risk area determined after death. The risk area during left anterior descending occlusion at a standoff of 2 to 3 cm was significantly larger at a 1500-ms pulsing interval (6.5 +/- 2.6 cm(2) for 2- to 3-cm standoff versus 3.7 +/- 1.4 cm(2) for 4- to 5-cm standoff; P =.01). The spatial extent at the 2- to 3-cm standoff more closely approximated risk area measured with Monastral Blue (7.8 +/- 2.7 cm(2)). Myocardial perfusion abnormalities during peak dobutamine stress were significantly smaller with the 4- to 5-cm standoff and undetectable in 4 of the 5 dogs. We conclude that ultrasound beam attenuation can reduce the size of a myocardial perfusion abnormality observed with intermittent harmonic imaging during a continuous infusion of microbubbles. This may reduce the sensitivity of this technique when transthoracic imaging is used.

Albumins↗

PubMed1999

Mixed hematopoietic chimerism prevents allograft vasculopathy.

BACKGROUND: Mixed hematopoietic chimerism has been shown to induce long-term acceptance of transplant organs. We determined whether mixed chimerism prevented allograft vasculopathy, using the rat aortic allograft model. METHODS: Mixed chimeras were prepared by reconstituting lethally irradiated (1100 cGy) WF rats with a mixture of T-cell depleted (TCD) syngeneic (WF) plus TCD allogeneic (ACI) bone marrow. Donor-specific (ACI) or third-party (F344) aortic grafts were transplanted into mixed chimeric animals 1 to 2 months after bone marrow reconstitution. No immunosuppressive drugs were administered. At 30 days postoperatively, aortic allografts were harvested for histology and measurement of cytokine mRNA by semiquantitative RT-PCR. Some aortic grafts were harvested at 90 and 180 days after transplantation for histological analysis. The degree of intimal hyperplasia and cytokine gene expression were compared among 4 groups: I (syngeneic; ACI donors to ACI recipients), II (allografts; ACI to WF), III (donor specific; ACI donor to chimeras) and IV (third-party; F344 to chimeras). RESULTS: There was no difference in the degree of intimal hyperplasia (IH) between groups I and III. Groups II and IV had significantly more IH than group I. Compared to group I, levels of mRNA for IFN-y, IL-2, IL-10 and iNOS in groups II and IV were higher, while there was no difference in mRNA levels between group I and III. CONCLUSIONS: These data suggest that mixed chimerism prevents allograft vasculopathy. Mixed chimerism holds great promise in clinical transplantation as a means to prevent allograft vasculopathy.

Animals↗

PubMed1999

Fundamental relationships between the composition of pluronic block copolymers and their hypersensitization effect in MDR cancer cells.

PURPOSE: Previous studies have demonstrated that Pluronic block copolymers hypersensitize multiple drug resistant (MDR) cancer cells, drastically increasing the cytotoxic effects of anthracyclines and other anticancer cytotoxics in these cells. This work evaluates the dose dependent effects of these polymers on (i) doxorubicin (Dox) cytotoxicity and (ii) cellular accumulation of P-glycoprotein probe, rhodamine 123 (R123) in MDR cancer cells. METHODS: Dox cytotoxicity and R123 accumulation studies are performed on monolayers of drug-sensitive (KB, MCF-7, Aux-B1) and MDR (KBv, MCF-7/ADR, CHrC5) cells. RESULTS: Both tests reveal strong effects of Pluronic copolymers observed at concentrations below the critical micelle concentration (CMC) and suggest that these effects are due to the copolymer single chains ("unimers"). Using block copolymers with various lengths of hydrophobic propylene oxide (PO) and hydrophilic ethylene oxide (EO) segments these studies suggest that the potency of Pluronic unimers in MDR cells increases with elevation of the hydrophobicity of their molecule. Optimization of Pluronic composition in R123 accumulation and Dox cytotoxicity studies reveals that Pluronic copolymers with intermediate lengths of PO chains and relatively short EO segments have the highest net efficacy in MDR cells. CONCLUSIONS: The relationship between the structure of Pluronic block copolymers and their biological response modifying effects in MDR cells is useful for determining formulations with maximal efficacy with respect to MDR tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers.

PURPOSE: Previous studies demonstrated that inhibition of P glycoprotein (P-gp) by Pluronic P85 (P85) block copolymer increases apical (AP) to basolateral (BL) transport of rhodamine 123 (R123) in the polarized monolayers of bovine brain microvessel endothelial cells (BBMEC) and Caco-2 cells. The present work examines the effects of P85 on the transport of fluorescein (Flu), doxorubicin (Dox), etoposide (Et), taxol (Tax), 3'-azido-3'-deoxythymidine (AZT), valproic acid (VPA) and loperamide (Lo) using BBMEC and Caco-2 monolayers as in vitro models of the blood brain barrier and intestinal epithelium respectively. METHODS: Drug permeability studies were performed on the confluent BBMEC and Caco-2 cell monolayers mounted in Side-Bi-Side diffusion cells. RESULTS: Exposure of the cells to P85 significantly enhanced AP to BL permeability coefficients of Flu, Tax, Dox and AZT in both cell models. Further, P85 enhanced AP to BL transport of Et, VPA and Lo in Caco-2 monolayers. No changes in the permeability coefficients of the paracellular marker mannitol were observed in the presence of the copolymer. CONCLUSIONS: P85 increases AP to BL permeability in BBMEC and Caco-2 monolayers with respect to a broad panel of structurally diverse compounds, that were previously shown to be affected by P-gp and/ or multidrug resistance associated protein (MRP) efflux systems. Broad specificity of the block copolymer effects with respect to drugs and efflux systems appears to be a valuable property in view of developing pharmaceutical formulations to increase drug accumulation in selected organs and overcome both acquired and intrinsic drug resistance that limits the effectiveness of many chemotherapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

SDS-induced conformational changes and inactivation of the bacterial chaperonin GroEL.

The inactivation and conformational changes of the bacterial chaperonin GroEL have been studied in SDS solutions with different concentrations. The results show that increasing the SDS concentration caused the intrinsic fluorescence emission intensity to increase and the emission peak to slightly blue-shift, indicating that increasing the SDS concentration can cause the hydrophobic surface to be slightly buried. The changes in the ANS-binding fluorescence with increasing SDS concentration also showed that the GroEL hydrophobic surface decreased. At low SDS concentrations, less than 0.3 mM, the GroEL ATPase activity increased with increasing SDS concentration. Increasing the SDS concentration beyond 0.3 mM caused the GroEL ATPase activity to quickly decrease. At high SDS concentrations, above 0.8 mM, the residual GroEL ATPase activity was less than 10% of the original activity, but the GroEL molecule maintained its native conformation (as indicated by the exposure of buried thiol groups, electrophoresis, and changes of CD spectra). The above results suggest that the conformational changes of the active site result in the inactivation of the ATPase even though the GroEL molecule does not markedly unfold at low SDS concentrations.

Adenosine Triphosphatases↗

PubMed1999

A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes.

Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer. This has made the cytoplasmic expression system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter. The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes. For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria. Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex. The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induces higher, more sustained levels (> 7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C- or with the nuclear expression system pCMV-CAT. This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.

Bacteriophage T7↗

PubMed1999

Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection.

Intravenous gene delivery via cationic lipidic vectors gives systemic gene expression particularly in the lung. In order to understand the mechanism of intravenous lipofection, a systematic study was performed to investigate the interactions of lipidic vectors with mouse serum emphasizing how serum affects the biophysical and biological properties of vectors of different lipid compositions. Results from this study showed that lipidic vectors underwent dynamic changes in their characteristics after exposure to serum. Addition of lipidic vectors into serum resulted in an immediate aggregation of vectors. Prolonged incubation of lipidic vectors with serum led to vector disintegration as shown in turbidity study, sucrose-gradient centrifugation analysis and fluorescence resonance energy transfer (FRET) study. Vector disintegration was associated with DNA release and degradation as shown in EtBr intercalation assay and DNA digestion study. Serum-induced disintegration of vectors is a general phenomenon for all cationic lipidic vectors tested in this study. Yet, vectors of different lipid compositions vary greatly in the rate of disintegration. There is an inverse correlation between the disintegration rate of lipidic vectors and their in vivo transfection efficiency. Vectors with a rapid rate of disintegration such as those containing dioleoyl-phosphatidylethanolamine (DOPE) poorly stayed in the lung and were barely active in transfecting cells. In contrast, cholesterol-containing vectors that had a rapid aggregation and a slow disintegration were highly efficient in transfecting cells in vivo. The results of this study explain why cationic lipidic vectors of different lipid compositions have a dramatic difference in their in vivo transfection efficiency. These results also suggest that the study of the interactions of lipidic vectors with serum may serve as a predictive model for the in vivo efficiency of a lipidic vector. Further study of the numerous interactions of lipidic vectors with serum might lead to the development of a vector which can deliver a gene to target cells in a tissue-specific manner.

Animals↗