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Biomedical subjects

S M Sullivan

Publications and source records attributed to S M Sullivan.

At least 19 recordsLinked to original sources
PubMed2006

Development and characterization of a synthetic promoter for selective expression in proliferating endothelial cells.

BACKGROUND: Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues. METHODS: Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter. RESULTS: The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues. CONCLUSIONS: These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors.

Animals↗
PubMed2006

Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) are essential for normal ocular development and are expressed in numerous ocular cell types including lens epithelial cells, retinal pigment epithelial cells, Müller cells and endothelial cells. Endothelial cell proliferation is a common feature of proliferative retinopathies and involves abnormal growth of blood vessels within and on the surface of the retina. In an effort to inhibit the formation of these aberrant blood vessels, we cloned an IGF-IR ribozyme into an expression vector that limits expression of the ribozyme to proliferating endothelial cells. An endothelin enhancer and Cdc6 promoter chimera drives expression of the IGF-IR ribozyme. This promoter limited retinal expression of the reporter gene to proliferating endothelial cells in two mouse models of proliferative retinopathy. In addition, expression of the IGF-IR ribozyme by this promoter inhibited aberrant retinal angiogenesis in both models while preserving normal vessels. These results demonstrate the feasibility of IGF-IR ribozyme expression in a selective manner for safer treatment of abnormal angiogenesis associated with retinopathy.

Animals↗
PubMed2005

Biological evaluation of penetration domain and killing domain peptides.

BACKGROUND: Cancer gene therapy must impact the majority of cells to be effective. Current gene delivery systems are unable to achieve sufficient transfer efficiency to the tumor cells. Cell killing can be dramatically increased through a bystander effect. Modeling the gene product with synthetic peptides can identify key elements for creating cell killing through a bystander effect. METHODS: Fluorescent labeled peptides were used for uptake kinetic studies and determination of intracellular localization in human glioblastoma cell lines, rat glioma cells lines and pressurized rat cerebral arteries. The degree of cell killing was assayed using propidium iodide coupled with fluorescence-activated cell sorting (FACS) analysis. RESULTS: Peptides derived from HIV Tat and Drosophila antennapedia homeodomain were taken up by all tumor and primary cells. Attachment of an Mdm-2-binding domain derived from P14(ARF) resulted in cell killing and was independent of domain orientation. Uptake kinetics showed rapid uptake for both tumor and primary cells equilibrating with the external media within 10 min. Intraluminal or extraluminal administration of peptides into pressurized cerebral arteries showed a lack of extravasation across the subbasement lamina. Assay of biological activity following intraluminal administration showed selective suppression of response to vasodilation with no effect on response by smooth muscle cells. CONCLUSIONS: The results from these studies identified: (1) a cell trafficking domain and a cytotoxic domain for killing brain tumor cells; (2) that cell killing was independent of the domain orientations with regard to the cell trafficking domain being at the C-terminus or N-terminus; and (3) that the dual domain peptide can also be taken up by endothelial cells as shown by the cerebral artery studies. Hence, localized expression of the cytotoxic gene has the potential to not only kill brain tumor cells, but also tumor endothelium, thus further increasing the effectiveness of the therapy.

Amino Acid Sequence↗
PubMed2004

Peptide-mediated gene transfer of cationic lipid/plasmid DNA complexes to endothelial cells.

The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (approximately 1 x 10(-7) M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.

Cations↗
PubMed2004

DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1.

Cell Adhesion Molecules↗
PubMed2004

The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase.

CgtA(E)/Obg(E)/YhbZ is an Escherichia coli guanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtA(E) with guanine nucleotides and show that its properties are similar to those of the Caulobacter crescentus homolog CgtA(C). CgtA(E) binds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtA(E) is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtA(E) copurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtA(E) and SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtA(E) is involved in the SpoT-mediated stress response.

Bacterial Proteins↗
PubMed2001

Measurement of one-bond 1H-13C, couplings in backbone-labelled proteins.

NMR dipole-dipole couplings between protein backbone nuclei (1H(alpha), 13C(alpha), 15N, 1H(N), 13C') offer enormous scope for the rapid determination of protein global folds. Here, we show that measurement of one-bond splittings in the protein backbone is facilitated by use of protein that is selectively isotopically enriched only in the backbone atoms. In particular, 1H(alpha)-13C(alpha) couplings can be measured simply and with high sensitivity by use of conventional heteronuclear single quantum correlation (HSQC) techniques.

Amino Acids↗
PubMed2001

Bone volume collected from dental implant sites during osteotomy.

PURPOSE: Little research has been done to determine the amount of bone harvested from implant site preparations using an inline bone collector. This study looked at the amount of bone that can be harvested from common dental implant osteotomies. PATIENTS AND METHODS: A total of 24 implants were placed in 9 patients over a 3-month period. Implant size ranged from 3.75 x 13 mm to 4.75 x 13 mm. Nine implants were placed in the maxilla, and 15 implants were placed in the mandible. Seven patients were female, and 2 patients were male. The patient age ranged from 27 to 72 years. Four patients had implants placed within 5 years after tooth extraction, and 5 patients had implants placed 5 years after tooth extraction: an analysis of variance was used to determine if there were statistical differences between maxilla versus mandible, male versus female, and edentulism less than or greater than 5 years. RESULTS: The average bone volume from the 24 osteotomies was 0.195 +/- 0.099 mL. The average osteotomy site measured 4.02 x 12.90 mm. There were no statistical differences noted among maxilla and mandible, gender, or time of edentulism. CONCLUSIONS: When using an inline bone collector to harvest implant osteotomy sites, an average of 0.195 mL of bone can be obtained from a site approximately 4.0 x 13 mm. This bone can often be combined with a xenograft or alloplastic material to provide extra bulk to fill peri-implant defects. When multiple implant sites are prepared, often sufficient bone can be obtained with the bone collector alone.

Adult↗
PubMed2001

The use of a resorbable plating system for treatment of craniosynostosis.

PURPOSE: The purpose of this study was to evaluate the effectiveness of a resorbable plating system (Lactosorb; Walter Lorenz Surgical, Inc, Jacksonville, FL) as a fixation method in the treatment of craniosynostosis. PATIENTS AND METHODS: Ten children with 15 affected sutures underwent craniotomies ranging from release of 1 suture to total calvarial reconstruction. The 1.5 Lactosorb plating system was used as the method of fixation in all cases. Patients were evaluated clinically and with computed tomography scans before discharge postoperatively, and at the 3-, 6-, and 9-month intervals. RESULTS: In all 10 cases there was no evidence of neosynostosis, malposed osseous segments, or restriction of growth or calvarial expansion. In addition, none of the complications seen with more traditional techniques were evident in these patients. CONCLUSION: Resorbable plating systems provide a viable alternative to the more traditional fixation techniques that have been used to treat craniosynostosis and craniofacial dysostosis.

Absorbable Implants↗
PubMed2000

Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium.

Efficient encapsulation of DNA plasmids inside small, neutral liposomes composed of 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), DOPC/DOPE (1,2-dioleoyl-sn-phosphatidylethanolamine) (1:1) and DOPC/DOPE/cholesterol (1:1:1) was achieved by the addition of ethanol and calcium chloride to an aqueous mixture of small unilamellar vesicles (SUVs) and plasmid. Following dialysis against low-salt buffer, the neutral lipid complexes (NLCs) had average effective diameters less than 200 nm and encapsulated up to 80% of the DNA. Optimum Ca(2+) and ethanol concentrations for each lipid mixture were determined by statistically designed experiments and mathematical modeling of trapping efficiency. NLCs are unilamellar, have neutral surface potentials, and retain entrapped DNA at pH 4.0 and in serum at 37 degrees C. The circulation and clearance properties of the complexes following intravenous administration in mice are similar to empty neutral liposomes, and the toxicity of NLCs are expected to be significantly reduced compared to other non-viral gene-delivery systems. The NLC encapsulation method, if it can be combined with effective targeting and endosome-release technologies to achieve efficient and tissue-specific transfection, may represent an important alternative to current systemic gene therapy approaches.

Animals↗
PubMed2000

Bacterial division: Finding the dividing line.

Division of a cell - whether eukaryotic or prokaryotic - requires accurate spatial coordination. Recent work on the bacterium Escherichia coli has shown that correct placement of the cell division site at the midcell position occurs by a combination of selection against potential polar sites and selection of the midcell site.

Adenosine Triphosphatases↗
PubMed2000

Bacterial sporulation: pole-to-pole protein oscillation.

Sporulating bacteria need to temporally coordinate DNA replication, chromosome partitioning and sporulation initiation. Recent work has shown that one aspect of this coordination lies with the interdependent subcellular localization of two proteins, Spo0J and Soj, and in the Spo0J-dependent spatial oscillation of Soj.

Bacillus subtilis↗
PubMed2000

Cationic lipid-based delivery system for systemic cancer gene therapy.

A cationic lipid-based gene delivery system composed of N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 microg of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.

Animals↗
PubMed2000

Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration.

The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.

Animals↗
PubMed2000

Analysis of guanine nucleotide binding and exchange kinetics of the Escherichia coli GTPase Era.

Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3'-O-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (K(D)) for mGDP and mGTP (0.61 +/- 0. 12 microgM and 3.6 +/- 0.80 microM, respectively) are similar to those of the unmodified nucleotides. The single turnover rates for mGTP hydrolysis by Era were 3.1 +/- 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl(2) and 5.6 +/- 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl(2). Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg(2+). We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.

Escherichia coli↗
PubMed2000

Targeted gene delivery: a two-pronged approach.

The success of gene therapy relies on the ability of gene delivery systems to selectively deliver therapeutic genes to a sufficient number of target cells yielding expression levels that impact the diseased state. The gene delivery systems can be divided into two classes: viral and nonviral (or plasmid DNA-based). The present gene delivery technology being used in clinics today can be considered first generation, in that they possess the ability to transfect or infect target cells through their inherent chemical, biochemical, and molecular biological properties. Relying on these sole properties, however, limits therapeutic applications. Expansion of therapeutic applications or increased effectiveness of current therapies can be achieved by increasing the number of cells and cell types susceptible to gene transfer. This can be achieved through physical targeting and molecular biological targeting. Physical targeting relies on the attachment to the delivery vehicle of ligands that bind to cell surface receptors unique to the target cells. Molecular biological targeting refers to selective expression of the therapeutic gene by the target cell through the use of selective promoters. Selective expression can be further achieved by the use of expression systems controlled by extrinsic induction molecules. This review will describe in detail the advances that have been made in each of these areas of gene targeting.

Animals↗
PubMed1999

Practice differences between male and female oral and maxillofacial surgeons: survey results and analysis.

PURPOSE: This study describes the personal and practice characteristics of oral and maxillofacial surgeons, with an emphasis on gender differences. Potential explanations for differences found are offered. MATERIALS AND METHODS: A 39-item questionnaire was designed to address areas of suspected differences between male and female oral and maxillofacial surgeons. It included items regarding training, certification, practice type and location, time spent working, practice composition, and personal characteristics. Identical questionnaires were sent to all of the 130 female oral and maxillofacial surgeons (OMSs) registered with AAOMS, as well as to 264 randomly sampled male OMSs. RESULTS: Of the 192 responses received, 109 were from male surgeons, and 83 were from females, 56.8% versus 43.2% of the total. For men, the response rate was 41.3%, whereas the rate was 68.3% for the women; overall, 48.7% of the 394 oral surgeons responded. Profiles of the "average" male and female OMS showed similarities with regard to race, training, and reasons for choosing an OMS career. Age, marital status, number of children, physical characteristics, and use of time outside of work indicated some significant personal differences, as did practice characteristics, including income, number of years in practice, hours spent working, and number of patients seen. Clear gender-based trends were found regarding opinions of physical and mechanical disadvantages, with women more strongly denying such problems. A variety of both positive and negative viewpoints were elicited by the free comment section. CONCLUSIONS: Although male and female OMSs are similar with regard to most variables investigated, some significant differences do exist.

Adult↗