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Biomedical subjects

R W Graham

Publications and source records attributed to R W Graham.

At least 19 recordsLinked to original sources
PubMed2000

Activation of host antitumoral responses by cationic lipid/DNA complexes.

A model of lipoplex-induced peritonitis was used to characterize the inflammatory response to cationic lipid:DNA lipoplexes with respect to activation of host antitumoral effector mechanisms. Three different cationic lipids were used in these studies: N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-[2,3-dioleoyloxylpropyl)-N,N,N-trimethylammonium chloride (DOTAP), and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA). The DODAC and DOTMA lipoplexes exhibited similar transfection properties in vitro, whereas the DOTAP lipoplexes transfected quite poorly in all cell lines tested. Intraperitoneal injection of cationic lipoplexes into immunocompetent mice resulted in a profound infiltration of inflammatory cells, secretion of interferon-gamma, and increased natural killer activity within the peritoneal cavity. Both DODAC and DOTMA lipoplexes produced similar inflammatory responses, lasting at least 5 days. The inflammation induced by DOTAP lipoplexes peaked by day 3 and resolved to near-control levels by day 5. These data indicate that although cationic lipid DNA complexes may differ in their inflammatory properties, the natural killer activation and interferon-gamma secretion that follow lipoplex administration should provide a functional adjuvant for cancer gene therapies that benefit from immunostimulation.

Animals↗
PubMed2000

Stabilized plasmid-lipid particles for systemic gene therapy.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.

Animals↗
PubMed1999

Stabilized plasmid-lipid particles: construction and characterization.

A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.

Animals↗
PubMed1999

Cationic lipid-mediated transfection of cells in culture requires mitotic activity.

Cationic lipid-based delivery systems such as lipoplexes or stabilized plasmid-lipid particles (SPLP) represent a safer alternative to viral systems for gene therapy applications. We studied the impact of cell cycle status on the efficiency of transfection of human ovarian carcinoma tumor cells using two cationic-lipid based delivery systems. Cells arrested in the G1 phase of the cell cycle by treatment with aphidicolin were compared with an asynchronous dividing population of cells. Treatment of the cells with aphidicolin had no effect on the rate of internalization of the lipid formulated DNA or on the level of gene expression observable in stably transfected cells. However, cells treated with aphidicolin exhibited 20-fold lower reporter gene activity than asynchronous control cells upon incubation with lipoplexes. When cells arrested in the G1 phase were allowed to proceed through the cell cycle in the presence of the lipoplex or SPLP, transgene expression was found to coincide with the transition of cells from the G2/M phase into the G1 phase of the subsequent cell cycle. In addition, higher levels of reporter gene expression were observed when the cells were incubated with lipoplexes or SPLP during, or just before, mitosis. These results suggest that it may be possible to augment cationic lipid-mediated transfection by manipulating the cell cycle status of the target cells.

Cations↗
PubMed1999

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer.

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.

Blotting, Southern↗
PubMed1999

Progress towards a synthetic virus for systemic gene therapy.

Here we discuss recent progress in non-viral gene therapy with an emphasis on developments which specifically attempt to address the limitations of current systems and their inability to overcome the first barrier to systemic gene delivery, delivery to the disease site and the target cell. Other issues associated with the efficiency of transfection, such as intracellular delivery, the endosomal release of vectors internalized through endocytosis and nuclear delivery are also discussed.

Animals↗
PubMed1995

Formation of novel hydrophobic complexes between cationic lipids and plasmid DNA.

An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗
PubMed1995

The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli.

Plasmids pTugA and pTugAS, designed for expression of cloned genes in Escherichia coli, possess the features of high-level inducible transcription, enhanced RNA translation, portability, high copy number, stability and versatility. In addition, pTugAS can be used to produce fusion proteins comprising a target protein and a cellulose-binding domain. Such fusion proteins can be purified in a single step by affinity chromatography on cellulose. Expression of two model gene fusions using the pTug plasmids resulted in yields of 500 mg of intracellular and 250 mg of extracellular recombinant protein per liter.

Alkaline Phosphatase↗
PubMed1995

A portable regulatory element directs specific expression of the Caenorhabditis elegans ubiquitin gene ubq-2 in the somatic gonad.

The Caenorhabditis elegans ubq-2 gene encodes a fusion of ubiquitin and a 52-amino-acid ribosomal protein. This single copy gene is both cis- and trans-spliced. It is expressed in all life stages of the worm and its transcript abundance is unaffected by heat stress. Transgenic analysis shows that expression of ubq-2 is regulated by an upstream promoter and a downstream element. The downstream element is required for ubq-2 promoter activity in embryos and in cells of the somatic gonad, including the distal tip cells, sheath cells, spermathecal cells, and cells of the uterus. The gonad-specific activity of the downstream regulator is transferable to a stress gene promoter such that heat-inducible expression of the transgene occurs in the somatic gonad. Stress-inducible beta-galactosidase expression in the gonad does not occur in all life stages, but is initiated late in the second or early in the third larval stage, when differentiation of gonadal tissues begins. Expression of a beta-galactosidase fusion protein from constructs containing the downstream ubq-2 regulator causes abnormalities of the gonad and embryonic lethality. Gonad abnormalities include arrested development and general disorganization. These abnormalities may be related to the overexpression of ubiquitin in the gonad.

Amino Acid Sequence↗
PubMed1995

Laparoscopic extraperitoneal bladder neck suspension (LEBNS) for stress urinary incontinence.

Seventy-nine patients of bladder neck suspension using an extraperitoneal variation of laparoscopic surgery were performed for the treatment of stress urinary incontinence. Using a balloon dissector the anterior vesical pelvic space is secured. The bladder neck suspension similar to the Burch operation was performed through a laparoscopic procedure. Symptoms of patients were assessed preoperatively and at one and six months following surgery. Operative times and complications were also evaluated. Success rate was 89.8% at six months. Complications such as bladder perforations were observed. Laparoscopic extraperitoneal bladder neck suspension-(LEBNS) is a viable option to the conventional methods of suspension, it has definite cosmetic advantages, is devoid of intraperitoneal dissection and adhesion, and has a comparable success rate.

Adult↗
PubMed1993

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.

Base Sequence↗
PubMed1989

Differential regulation of closely related members of the hsp16 gene family in Caenorhabditis elegans.

The heat-inducible genes encoding 16-kD heat shock polypeptides in Caenorhabditis elegans are found at two separate loci, one containing the 16-1 and 16-48 genes (locus A), and the other, the 16-2 and 16-41 genes (locus B). Despite the highly conserved structures of these genes and their promoters, the B locus produces up to sevenfold more mRNA during heat induction than does the A locus. Since there are two copies of the 16-1 and 16-48 genes at the A locus, the discrepancy in mRNA production is actually as high as 14:1 on a per gene basis. Measurements of the rate of hsp16 mRNA decay during recovery from a heat shock suggest that this difference is not caused by differential mRNA stability; furthermore, nuclear runon experiments yield rates of transcription for the 16-1/48 locus that are approximately threefold higher than those from the 16-2/41 locus. The higher levels of mRNA from the 16-2/41 locus, particularly at longer induction times, seem to be due to a marked difference in the temporal pattern of mRNA production from the two loci. While both loci are transiently activated by a heat shock, the 16-1 and 16-48 genes of the A locus are down-regulated to a lower transcription rate sooner than the genes from the B locus.

Animals↗
PubMed1989

UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed.

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.

Animals↗
PubMed1989

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans.

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Adult day care: how families of the dementia patient respond.

1. The experience of caring for a family member with dementia in the home is a physical, emotional, and financial strain for the majority of caregivers. 2. The availability of community as well as family supports are variables which will influence how well a caregiver will cope. 3. Though the use of adult day care did not significantly decrease the reported stress for caregivers as a group, some did experience less strain. 4. Caregiver response to adult day care is variable and complex. Gerontological nurses who counsel caregivers should help them be realistic about their expectations for adult day care.

Aged↗
PubMed1988

Maturation of the major ubiquitin gene transcript in Caenorhabditis elegans involves the acquisition of a trans-spliced leader.

Recently, studies on the 5'-ends of actin mRNAs in the nematode, Caenorhabditis elegans, have demonstrated that three of the four mature actin transcripts contain a 22-nucleotide leader sequence which is acquired by trans-splicing from a novel 100-nucleotide RNA. In the course of our studies of the ubiquitin genes in C. elegans (R. W. Graham and E. P. M. Candido, manuscript in preparation) we were led to the possibility that the major ubiquitin transcript in this organism might also contain a trans-spliced leader sequence. The 5'-noncoding region of the major ubiquitin gene (UbiA) contains a 3'-splice consensus sequence six nucleotides upstream of the initiation codon; however, sequencing of a further 1.6 kilobases upstream failed to reveal the existence of any potential 5'-splice sites in the correct relationship to known promoter elements. Furthermore, S1 and Northern blot analyses using probes complementary to upstream regions failed to detect the ubiquitin transcript. Primer extension experiments on total RNA using an oligonucleotide which hybridized to the ubiquitin transcript downstream of the 3'-splice site demonstrated the existence of a 22-nucleotide leader sequence not present in the ubiquitin gene 5'-region. Thus we show here that the 2500-nucleotide mRNA encoding a polyubiquitin precursor protein contains a 22-nucleotide leader sequence identical to that found in several C. elegans actin mRNAs and which is most likely acquired by a trans-splicing mechanism. In addition we have localized the site of transcript initiation for UbiA by S1 nuclease mapping.

Amino Acid Sequence↗