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Biomedical subjects

M E Hogan

Publications and source records attributed to M E Hogan.

At least 37 records · Page 2Linked to original sources
PubMed1997

Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.

Mice harboring a targeted disruption of the epidermal growth factor receptor (EGFR) allele exhibit a severely disorganized hair follicle phenotype, fuzzy coat, and systemic disease resulting in death before 3 weeks. This skin phenotype was reproduced in whole skin grafts and in grafts of EGFR null hair follicle buds onto nude mice, providing a model to evaluate the natural evolution of skin lacking the EGFR. Hair follicles in grafts of null skin did not progress from anagen to telogen and scanning electron micrografts revealed wavy, flattened hair fibers with cuticular abnormalities. Many of the EGFR null hair follicles in the grafted skin were consumed by an inflammatory reaction resulting in complete hair loss in 67% of the grafts by 10 weeks. Localization of follicular differentiation markers including keratin 6, transglutaminase, and the hair keratins mHa2 and hacl-1 revealed a pattern of premature differentiation within the null hair follicles. In intact EGFR null mice, proliferation in the interfollicular epidermis, but not hair follicles, was greatly decreased in the absence of EGFR. In contrast, grafting of EGFR null skin resulted in a hyperplastic response in the epidermis that did not resolve even after 10 weeks, although the wound-induced hyperplasia in EGFR wild-type grafts had resolved within 3 to 4 weeks. Thus, epithelial expression of the EGFR has complex functions in the skin. It is important in delaying follicular differentiation, may serve to protect the hair follicle from immunological reactions, and modifies both normal and wound-induced epidermal proliferation but seems dispensable for follicular proliferation.

Age Factors↗
PubMed1997

Harlequin ichthyosis (ichq): a juvenile lethal mouse mutation with ichthyosiform dermatitis.

The harlequin ichthyosis (ichq) mouse mutation arose spontaneously in 1989 in a colony of BALB/cJ mice at The Jackson Laboratory. Affected mice developed thick skin due to formation of compact, orthokeratotic scales that fractured over articular surfaces, secondary to bending. Harlequin ichthyosis mice on the inbred BALB/cJ background died between 9 and 12 days of age. Onset of the clinical phenotype corresponded with emergence of hair fibers from follicles at 5 days of age. There was marked proliferation of the root sheaths of anagen hair follicles, limited to the region within the dermis. Sebaceous glands were present but small compared with those of littermate controls. Emerging hair fibers were surrounded by a thick, compact sheath of cornified cells. Mutant skin contained large mitochondria with lamellar-shaped, electron-dense structures at the ultrastructural level. Keratohyalin granules were smaller and less pleomorphic than those in control mice. Lamellar bodies were not evident in either mutant or littermate control mice. Using a panel of antibodies to evaluate changes in keratinocyte differentiation, mouse-specific keratin 6 was overexpressed in the suprabasilar, hyperplastic epidermis. Loricrin expression, within the cytoplasm of cells in the stratum granulosum, decreased rapidly postmortem, unlike that in normal mice where it was stable for over 24 hours postmortem. Filaggrin expression, within granules of cells in the stratum granulosum, was prominent, corresponding to hypergranulosis evident by light microscopy in mutant mouse skin. Skin grafts from harlequin ichthyosis mice grafted onto immunodeficient nude mice maintained the phenotype for the 10-week observation period. The mutant gene locus mapped to the proximal end of mouse chromosome 19 and is inherited as a fully penetrant autosomal recessive gene. The harlequin ichthyosis mouse mutation is very similar to human type 2 harlequin ichthyosis for which it may be a good model.

Animals↗
PubMed1996

Microscopic DNA flexibility analysis. Probing the base composition and ion dependence of minor groove compression with an artificial dna bending agent.

We have used an artificial DNA bending agent to monitor the local flexibility of the DNA helix as a function of Mg2+ cation concentration, sequence, and temperature. A DNA bending agent was constructed from a pair of triple helix-forming oligonucleotides connected by a flexible polymeric linker, which, when the linker is short enough, causes a bend in a minor groove region separating the two sites of triple helix formation. The unique aspect of this system is that, since the bent region is not in direct contact with the linker or the triple helix-forming oligonucleotides, the free energy reflecting the bendability of the minor helix groove can be estimated from a comparison of binding affinity between the bent and unbent triple helices. A binding competition experiment and association and dissociation kinetic assays executed at 37 degrees C in the presence of 10 mM Mg2+ have revealed an extremely small difference in binding affinity between bent (50 degrees ) and straight triple helices, suggesting that DNA flexibility with respect to minor groove compression is extremely high and virtually independent of the sequence of the distorted duplex. This unexpectedly small difference in binding affinity was detected over the temperature range from 25 to 65 degrees C, and over a Mg2+ concentration range from 0.3 to 10 mM. Thus, these findings provide evidence that DNA bendability for minor groove compression is inherently high and independent of DNA sequence, temperature, or a 30-fold variation of Mg2+ ion concentration.

Binding, Competitive↗
PubMed1996

Triple helix-forming oligodeoxyribonucleotides targeted to the human tumor necrosis factor (TNF) gene inhibit TNF production and block the TNF-dependent growth of human glioblastoma tumor cells.

Synthetic oligodeoxyribonucleotides (ODNs) designed to selectively inhibit the transcription or translation of specific genes are being used to modulate the activity of the targeted gene. Because multiple copies of mRNA can be transcribed from one actively expressed gene, ODNs that target double-stranded DNA and form triple helices upon binding with the gene itself have an advantage over ODNs that target the gene product (mRNA) in an antisense fashion. For the present studies, we designed four different triple helix-forming phosphodiester ODNs (TFOs) targeted to the tumor necrosis factor (TNF) gene and examined their effect on production of TNF and on cellular growth of tumors in which TNF acts as an autocrine growth factor. The ODNs J-109-50 and J-108-57 were designed to interact with polypurine oligonucleotides corresponding to the binding sites for nuclear factors kB (-237 to -208) and Sp1 (-58 to -33), respectively; J111-51 was designed to interact with a polypurine oligonucleotide in the third intron (+1429 to +1456) of the TNF gene. To enhance the cellular penetration and prevent degradation by cellular nucleases, the TFOs were modified at their 3' ends by either a cholesterol side chain or a propanolamine blocking group. Treatment of the human promonocytic cell line THP-1 with TNF-TFOs at a nontoxic concentration (2 microM) reduced the production of TNF. All of the TNF-TFOs tested were effective, and control-irrelevant TFOs were ineffective in inhibiting TNF production. The activity of the most efficacious TNF-TFOs also correlated with a decrease in TNF mRNA as observed by using reverse transcriptase PCR assays. In several tumors in which TNF acts as an autocrine growth factor, we examined the antiproliferative activity of J111-51. We found that in the human glioblastoma tumor cell line U-251, TNF-induced growth was blocked by J111-51 in a dose-dependent manner. Thus, overall results demonstrate that oligonucleotides directed to the specific regions of TNF can be designed, which may have a potential in cancer therapy.

Animals↗
PubMed1996

The design of an agent to bend DNA.

An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70 degrees bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70 degrees bend was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.

Base Sequence↗
PubMed1996

High-efficiency triple-helix-mediated photo-cross-linking at a targeted site within a selectable mammalian gene.

Targeting damage to specific sites in the genome represents an attractive approach to manipulating gene function in mammalian cells. To test the applicability of triple-helix formation as a means for achieving precisely timed site-specific damage within a mammalian gene, a triplex-forming oligodeoxyribonucleotide (TFO) that binds with high affinity to a specific site within the hamster adenine phosphoribosyltransferase (APRT) gene was modified with the photochemically reactive psoralen derivative 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). The modified TFO, psorTFO1, bound with high affinity to a target site within intron 1 of the APRT gene. Upon irradiation, photomonoadducts (i.e., covalent adducts of psorTFO1 to one strand of the target duplex) were formed with high efficiency (approximately 50%). Introduction of 5'-TpA sequences (the preferred site for psoralen-induced photo-cross-links) at or near the triplex junction leads to increased efficiency of total photoadduct formation and to efficient formation of products that had the electrophoretic mobility on denaturing PAGE expected for three-stranded photo-cross-links (i.e., products containing psorTFO1 covalently linked to both strands of the duplex). Their identities as cross-links were verified by (1) identical electrophoretic mobility of products formed with either duplex strand radiolabeled and (2) coprecipitation of the radiolabeled duplex strand with its complementary biotinylated strand following denaturation. In addition, the cross-links were completely reversible upon irradiation at 254 nm, as expected for psoralen-mediated cross-links. The yield and distribution of photoadducts depended on 5'-TpA position. The most efficient photoadduct formation (approximately 90%) and photo-cross-link formation (approximately 90% of total photoadducts) were observed for a 5'-TpA adjacent to the triplex junction, with significant, but lower, cross-linking efficiency within three base pairs of the junction. Molecular models of the psoralen-conjugated triplex with its six-carbon linker suggested a simple explanation for this distance dependence: facile intercalation near the triplex/duplex junction, with increasing strain required for intercalation at more distant sites. These results indicate that psorTFO1 allows for DNA damage with high precision and high efficiency, and the likely proportion of monoadducts and cross-links can be estimated from the target sequence.

Adenine Phosphoribosyltransferase↗
PubMed1996

Solution structure of an antiparallel purine motif triplex containing a T.CG pyrimidine base triple.

BACKGROUND: Triplex formation is an approach of potential use in regulating and mapping of gene sequences. However, such applications have been limited to homogeneous sequences consisting of stretches of purines or pyrimidines. Understanding how heterogeneous duplexes are recognized by a third strand oligonucleotide at the atomic resolution level is an essential step toward broadening the application of triplex formation into biochemical and biomedical areas. RESULTS: The solution structure of an antiparallel triplex (RRY6) containing a site of inversion (i.e. a T within a homopurine stretch, forming a T.CG base triple) has been determined using NMR-restrained computations in the presence of explicit water. The results reveal that within the RRY6 triplex the conformation of the duplex is mostly B-like and that of the third strand exhibits significant variations in interbase separations and backbone torsion angles. A major displacement of the inversion site T sugar in a 5'-direction, accompanied by the tilt of the T base in T.CG, was observed. The T.CG base triple contains a single hydrogen bond between T O4 and the exposed C amino proton and is stabilized by a number of interstrand and sequential van der Waal contacts. The structural comparisons of RRY6 with two related triplexes indicate localized perturbation at the non-classical base triple site. Various triplexes contain sugars in the C2'-endo family and the global features of their duplexes are similar. CONCLUSIONS: This study provides valuable information concerning the molecular basis of the specific recognition of a Watson-Crick base paired C residue at the inversion sites in the antiparallel triplex and should lead to general rules for designing triplexes containing heterogeneous sequences.

Base Sequence↗
PubMed1996

Delivery of DNA-cationic liposome complexes by small-particle aerosol.

Aerosol delivery of gene therapy for treatment of lung diseases allows topical treatment of the airways with DNA concentrations not obtainable by systemic administration. We have investigated delivery of cationic liposomes complexed to plasmid DNA in a small particle aerosol. Plasmid cDNA-DMRIE/DOPE complexes were nebulized using either an Aerotech II or Puritan-Bennett 1600 (PB1600) nebulizer. Reservoir sampling showed that DNA-DMRIE/DOPE complexes were damaged to a significant degree during nebulization, such that activity of transfected gene was diminished. Of the nebulizers analyzed, DNA-DMRIE/DOPE complexes were more stable in the PB1600. The loss of effective transfection by DNA-DMRIE/DOPE, as detected by decreased reporter gene activity in A549 lung cells, was consistent with denaturation of the DMRIE/DOPE. In contrast, nebulized DNA-DOSPA/DOPE complexes retained complete ability to transfect. Adjustments to flow rate and reservoir volume of the PB1600 allowed a longer period of delivery of active DNA-DMRIE/DOPE particles. DNA-DMRIE/DOPE was radiolabeled with Technetium-99m (99mTc), nebulized, and the output captured in either an Andersen Sampler (AS) (Andersen, 1958) cascade impactor particle size analyzer or an all glass impinger. cDNA-cationic lipid complexes were detected in size ranges of 0.4-10 microns, with most particles found between 1-2 microns. Aerosol output was consistent from 0 to 5 min. These results show the feasibility of aerosol delivery of DNA-cationic lipids for the purposes of gene therapy to the lung.

Aerosols↗
PubMed1996

Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Antiviral Agents↗
PubMed1996

Lanceolate hair (lah): a recessive mouse mutation with alopecia and abnormal hair.

A new autosomal recessive mutation of the house mouse developed generalized alopecia associated with breakage of abnormal hair shafts. This mutation, named 'lanceolate hair' (symbol: lah), arose in a mutagenesis experiment using ethylnitrosourea. Hair shafts were short with a focal degeneration at the breakpoint characterized by a pronounced enlargement at the apex, resembling a lance head. Plucked hair fibers were 2.0 to 3.5 mm in length with a normal base, suggesting that there was a synchronized developmental defect. Histologic examination of anagen follicles revealed abnormal cornification of the matrix region with degeneration resulting in the focal hair shaft deformity. Catagen follicles showed pronounced follicular dystrophy but telogen follicles were almost normal. There was a marked, persistent thickening of the epidermis associated with a non-scarring, relatively non-inflammatory ichthyosiform dermatitis. These features are found in the Netherton's syndrome of the human, for which this mouse mutation may represent a model. The lah mutation has been localized to the centromeric end of mouse Chromosome 18.

Alopecia↗
PubMed1995

High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.

Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd = 7 x 10(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Phosphoribosyltransferase↗
PubMed1995

Triplex formation at the rat neu gene utilizing imidazole and 2'-deoxy-6-thioguanosine base substitutions.

Triplex-forming oligodeoxyribonucleotides (TFOs) can be designed so as to form antiparallel triple helices with duplex DNA by means of GGC and TAT or AAT base triplets, and these have been shown to be useful as sequence-specific DNA binding agents. Using TFOs targeted to the promoter region of the rat neu oncogene, it is shown here that substitution of an imidazole-nucleoside chimera at a single site in a neu specific TFO results in an increase in TFO binding affinity and specificity. This effect is discussed in terms of the stabilizing effect of local imidazole-TA triplet formation. It is also found that site-selective substitution of 2'-deoxy-6-thioguanosine for guanosine (S6-dG) in the TFO results in an increase in triplex formation in the presence of physiological levels of potassium ion. The utility and positioning of S6-dG base substitutions is discussed in the context of an intramolecular tetrad model.

Animals↗
PubMed1995

Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads.

An oligonucleotide (I100-15) composed of only deoxyguanosine and thymidine was able to inhibit human immunodeficiency virus type-1 (HIV-1) in culture assay systems. I100-15 did not block virus entry into cells but did reduce viral-specific transcripts. As assessed by NMR and polyacrylamide gel methods, I100-15 appears to form a structure in which two stacked guanosine tetrads are connected by three two-base long loops. Structure/activity experiments indicated that formation of intramolecular guanosine tetrads was necessary to achieve maximum antiviral activity. The single deoxyguanosine nucleotide present in each loop was found to be extremely important for the overall antiviral activity. The toxicity of I100-15 was determined to be well above the 50% effective dose (ED50) in culture which yielded a high therapeutic index (> 100). The addition of a cholesterol moiety to the 3' terminus of I100-15 (I100-23) reduced the ED50 value to less than 50 nM (from 0.12 microM for I100-15) and increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of the drug from infected cell cultures. The favorable therapeutic index of such molecules coupled with the prolonged suppression of HIV-1, suggest that such compounds further warrant investigation as potential therapeutic agents.

3T3 Cells↗
PubMed1995

Cutaneous ultrastructural features of the flaky skin (fsn) mouse mutation.

An autosomal recessive genetic disease with clinical and histopathological skin features resembling human psoriasis vulgaris occurs naturally in flaky skin mice (fsn/fsn). Affected mice are normal at birth, except for a hypochromic anemia. Subsequently, they develop hyperkeratotic plaques and acanthosis with elongation of rete ridges. Scanning electron microscopic examination revealed a greatly thickened epidermis, a sparsity of hairs and scale accumulations on the epidermal surface. Hair shafts had conspicuous pits, striations, and exophytic protrusions. Nails were bent at a 90 degrees angle with surface irregularities and accumulations of scale at the nail base. Transmission electron microscopic examination showed increased epidermal thickness, mitochondrial aberrations, and intraepidermal invasion by neutrophils. Keratohyalin abnormalities were detected using immunocytochemical staining for profilaggrin. At the dermal-epidermal junction, numerous macrophages and mast cells were seen in close proximity to focal dissolutions of the basement membrane. A high density of collagen fibers and cellular infiltrates were evident in the papillary dermis. This constellation of ultrastructural aberrations is typically found in psoriasis vulgaris and supports the theory that the flaky skin mouse mutation is a naturally occurring analog to one variety of human psoriasis vulgaris.

Anemia, Hypochromic↗
PubMed1994

Triplex formation at the rat neu oncogene promoter.

Current cancer chemotherapy treatments generally act by affecting rapidly growing malignant cells. Unfortunately, they are relatively nonspecific and thus have a tendency to affect other rapidly growing normal cells in a deleterious manner. Triplex-forming oligodeoxyribonucleotides (TFOs) promise to be a new class of sequence-specific DNA-binding drugs which will target malignancies at the transcriptional level. The formation of an intermolecular triplex (triple helix) has been shown to block the binding of transcription factors and repress transcription in genes such as c-myc and that encoding the epidermal growth factor receptor. The rat neu oncogene promoter contain promoter-enhancer elements which are purine/pyrimidine rich. These enhancer elements are amenable to targeting by TFOs. the human counterpart of rat neu, HER2, is often found to be amplified or overexpressed in a variety of malignancies, such as those of the breast, lungs, ovary, colon and stomach. TFOs may proved to be the basis of effective chemotherapy drugs for these cancers. TFO binding at the "GTG" element (5'GGTGGGGGGG) and at the 'GA' element (5'GGAGGAGGAGGG) has been characterized by gel mobility shift analysis and DNase 1 footprinting. Binding has been shown to occur at a Kd as low as 10(-8) M and has been shown to be sequence specific.

Animals↗
PubMed1994

Inhibition of transcription factor binding to the HER2 promoter by triplex-forming oligodeoxyribonucleotides.

We have identified a 28-bp homopurine/homopyrimidine sequence capable of triple helix (triplex) formation with G+T-rich oligodeoxyribonucleotides (oligos) within the critical proximal promoter of the HER2/neu/c-erbB2 (HER2) proto-oncogene. To investigate the possible therapeutic potential of triplex-forming oligos in HER2 overexpressing breast cancers, we have studied the ability of triplex formation to compete with and to inhibit the binding of a transcription factor to its consensus sequence at an adjacent site. Competition binding assays demonstrate that a triplex-forming oligo can inhibit transcription factor binding in a sequence-specific manner. Moreover, we find that the addition of both nucleotide and non-nucleotide 'tails' to triplex-forming oligos do not confer any enhancement of binding affinity, but provide additional inhibition of transcription factor binding, potentially by steric hindrance.

Base Sequence↗