Hyal2, where are you?
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Cartilage, Articular↗
Biomedical subjects
A D Miller
Publications and source records attributed to A D Miller.
Oncogenic transformation by the jaagsiekte sheep retrovirus envelope protein.
Retroviruses have played profound roles in our understanding of the genetic and molecular basis of cancer. Jaagsiekte sheep retrovirus (JSRV) is a simple retrovirus that causes contagious lung tumors in sheep, known as ovine pulmonary adenocarcinoma (OPA). Intriguingly, OPA resembles pulmonary adenocarcinoma in humans, and may provide a model for this frequent human cancer. Distinct from the classical mechanisms of retroviral oncogenesis by insertional activation of or virus capture of host oncogenes, the native envelope (Env) structural protein of JSRV is itself the active oncogene. A major pathway for Env transformation involves interaction of the Env cytoplasmic tail with as yet unidentified cellular adaptor(s), leading to the activation of PI3K/Akt and MAPK signaling cascades. Another potential mechanism involves the cell-entry receptor for JSRV, Hyaluronidase 2 (Hyal2), and the RON receptor tyrosine kinase, but the exact roles of these proteins in JSRV Env transformation remain to be better understood. Recently, a mouse model of lung cancer induced by JSRV Env has been developed, and the tumors in mice resemble those seen in sheep infected with JSRV and in humans. In this review, we summarize recent progress in our understanding the molecular mechanisms of oncogenic transformation by JSRV Env protein, and discuss the relevance to human lung cancer.
Animals↗
The complete mitochondrial genome of the mantid shrimp Harpiosquilla harpax, and a phylogenetic investigation of the Decapoda using mitochondrial sequences.
The complete mitochondrial DNA sequence was determined for the mantid shrimp Harpiosquilla harpax. These data demonstrate that the H. harpax mitochondrial genome is a 15,714 bp circular molecule and encodes the typical 37 metazoan mitochondrial genes (13 protein-coding, 22 tRNA, and two rRNA genes). The gene arrangement of H. harpax is consistent with that of the putative arthropod ancestral gene order as depicted by Limulus polyphemus. H. harpax was employed as an outgroup taxon for a phylogenetic investigation of the Decapoda using sequences from complete mitochondrial genomes. Whilst our results are largely in agreement with current taxonomic treatments, the relationships indicated among the reptantian decapods are novel. Our results provide strong statistical support for a sister-group relationship between the Achelata and the Astacida. These findings not only refute previous phylogenetic hypotheses, but also have serious implications for the interpretation of morphological and developmental evolution in the Decapoda. In addition we also investigated the effects of outgroup selection on the resolution of ingroup relationships. We found outgroup choice to significantly influence tree topology thus reinforcing the importance of appropriate outgroup selection in phylogenetic studies.
Animals↗
Midbrain muscarinic receptors modulate morphine-induced accumbal and striatal dopamine efflux in the rat.
Midbrain dopamine neurons are critical in mediating the rewarding effects of opiates in dependent rats, as well as modulating some manifestations of opiate withdrawal. Morphine is known to excite dopamine neurons and thereby facilitate forebrain dopamine transmission through inhibition of GABA neurons. Cholinergic neurons in the mesopontine laterodorsal and pedunculopontine tegmental nuclei provide the principal source of excitatory cholinergic input to ventral tegmental area and substantia nigra pars compacta dopamine-containing neurons, via actions on midbrain muscarinic and nicotinic acetylcholine receptors. The present study hypothesized that a reduction in tonic cholinergic input via blockade of midbrain muscarinic receptors would reduce the pharmacological effects of morphine on forebrain dopamine release. Using in vivo chronoamperometry, alterations in morphine-evoked dopamine efflux were monitored at stearate-graphite paste electrodes implanted unilaterally in the nucleus accumbens and striatum of urethane (1.5 g/kg) anesthetized rats, following the pharmacological inhibition of ventral tegmental area/substantia nigra pars compacta muscarinic receptors. The facilitatory effects of morphine (2.0 mg/kg, i.v.) on accumbens and striatal dopamine efflux were markedly reduced by prior infusion of the non-selective muscarinic receptor antagonist scopolamine (200 microg/microl) into the ventral tegmental area or substantia nigra pars compacta, respectively. These findings demonstrate that decreased activation of midbrain muscarinic receptors attenuates the excitatory effects of morphine on mesoaccumbens and nigrostriatal dopaminergic transmission.
Animals↗
Stimulation of innate immunity in newborn kids against Cryptosporidium parvum infection-challenge by intranasal/per-oral administration of liposomal formulation of N-L18-norAbu-GMDP adjuvant.
The effects of a liposomal preparation of lipophilic immunomodulator beta-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance to Cryptosporidium parvum infection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 microg, 200 microg, or 1000 microg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1 x 10(7) oocysts of C. parvum in 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 microg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P < 0.05). No cryptosporidia were found on the mucosal surface of treated kids by day 10, while the intestines of the control kids were still infected. All doses and routes of administration were judged effective with respect to suppression of cryptosporidia infections.
Adjuvants, Immunologic↗
Improved CNS tolerability following conversion from immediate- to extended-release carbamazepine.
OBJECTIVES: Tolerability of 'narrow therapeutic ratio' (NTR) antiepileptic drugs may improve with uniform drug delivery. We determined whether conversion from immediate-release carbamazepine (IR-CBZ) to extended-release carbamazepine (ER-CBZ) decreased the incidence of CNS side-effects associated with drug concentration oscillations. METHODS: We compared CNS side effects and seizure frequency for patients with partial-onset seizures (n = 61) treated with IR-CBZ for > or =1 year with conversion to ER-CBZ for > or =1 year. We compared tolerability findings with absorption variability of the formulations. RESULTS: Incidence of CNS side-effects decreased from 49% during IR-CBZ treatment to 20% following conversion to ER-CBZ. Patients also had improved tolerability of high doses (> or =1200 mg/day) during ER-CBZ treatment. Pharmacokinetic analysis showed absorption and drug concentration were much more variable for the immediate-release formulation. CONCLUSIONS: This study suggests that ER-CBZ formulations, with smoother drug delivery and less variable absorption, provide improved CNS tolerability compared with immediate-release formulations.
Adolescent↗
Identification of Hyal2 as the cell-surface receptor for jaagsiekte sheep retrovirus and ovine nasal adenocarcinoma virus.
Jaagsiekte sheep retrovirus (JSRV) and ovine nasal adenocarcinoma virus (ONAV) replicate in the airway and cause epithelial cell tumors through the activity of their envelope (Env) proteins. Identification of the receptor(s) that mediate cell entry by these viruses is crucial to understanding the oncogenic activity of Env and for the development of gene therapy vectors based on these viruses that are capable of targeting airway cells. To identify the viral receptor(s) and to further study the biology of JSRV and ONAV, we developed retroviral vectors containing Moloney murine leukemia virus components and the Env proteins of JSRV or ONAV. We used a new technique involving positional cloning by phenotypic mapping in radiation hybrid cells to identify and clone the human receptor for JSRV, Hyal2, which also serves as the receptor for ONAV. Hyal2 is a glycosylphosphatidylinositol-anchored cell-surface protein that has low hyaluronidase activity and is a member of a large family that includes sperm hyaluronidase (Spam) and serum hyaluronidase (Hyal1). Hyal2 is located in a region of human chromosome 3p21.3 that is often deleted in lung cancer, suggesting that it may be a tumor suppressor. However, its role in JSRV or ONAV tumorigenesis, if any, is still unclear. JSRV vectors are capable of transducing various human cells, and are being further evaluated for gene therapy purposes.
Adenocarcinoma↗
Electron solvation in two dimensions.
Ultrafast two-photon photoemission has been used to study electron solvation at two-dimensional metal/polar-adsorbate interfaces. The molecular motion that causes the excess electron solvation is manifested as a dynamic shift in the electronic energy. Although the initially excited electron is delocalized in the plane of the interface, interactions with the adsorbate can lead to its localization. A method for determining the spatial extent of the localized electron in the plane of the interface has been developed. This spatial extent was measured to be on the order of a single adsorbate molecule.
Journal Article↗
Excitotoxic lesions of the pedunculopontine differentially mediate morphine- and d-amphetamine-evoked striatal dopamine efflux and behaviors.
Cholinergic and glutamatergic cells in the pedunculopontine tegmental nucleus are a principal source of excitatory input to midbrain dopamine neurons projecting to the striatum. Disruption of these brainstem inputs has been shown to respectively enhance and reduce psychostimulant and opiate self-administration in rats. In the present study, d-amphetamine- and morphine-induced behaviors and dorsal striatal dopamine efflux, measured using in vivo chronoamperometry, were investigated 21 days after bilateral excitotoxic (ibotenate) lesions of the pedunculopontine in rats. Compared to sham-operated controls, pedunculopontine lesions enhanced stereotyped behaviors induced by a challenge injection of d-amphetamine (1.5 mg/kg, i.p.) to an extent that markedly interfered with the expression of locomotor behavior. A significant augmentation in striatal dopamine efflux was also observed in these lesioned animals under urethane anesthesia in response to a similar challenge injection of d-amphetamine (1.5 mg/kg, i.v.) 2 days following these behavioral observations. In direct contrast, pedunculopontine lesions in a separate group of rats significantly attenuated morphine-induced (2 mg/kg, i.p.) stereotyped activity, although no significant differences were observed in locomotion compared to sham-operated animals. Under urethane anesthesia, these lesions attenuated striatal dopamine efflux evoked by a similar challenge injection of morphine (2 mg/kg, i.v.). These findings indicate that the pedunculopontine differentially mediates the pharmacological actions of two diverse drugs of abuse on striatal dopamine neurotransmission and resultant behaviors. These results also imply that the pedunculopontine tegmental nucleus may serve as a major striatal-motor interface in the processing of salient environmental stimuli, and their incentive rewarding impact on dopamine-mediated behavioral responses.
Animals↗
Effects of laterodorsal tegmentum excitotoxic lesions on behavioral and dopamine responses evoked by morphine and d-amphetamine.
Cholinergic and glutamatergic projections from the laterodorsal tegmental nucleus (LDT) in the rat pons excite midbrain dopamine cells to directly modulate forebrain dopamine transmission. We show that LDT-lesioned rats express higher intensity stereotypy (including orofacial movements), and higher levels of accumbal dopamine release in response to d-amphetamine (1.5 mg/kg), as compared to sham-operated rats. In contrast, LDT-lesioned rats showed decreased stereotypy and attenuated accumbal dopamine efflux as compared to sham animals, in response to morphine (2.0 mg/kg). These results suggest that the LDT plays a critical role in mediating motoric and neurochemical effects of diverse drugs of abuse, and that the pharmacology of the drug may critically determine whether its efficacy will be enhanced or attenuated by alterations in LDT activity. We conclude that the LDT has functional connections with the nigrostriatal dopamine system to affect drug-evoked stereotypy, which has implications for motoric disorders that are characterized by nigrostriatal dysfunction.
Analgesics, Opioid↗
Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.
Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.
3T3 Cells↗
The molecular interactions of heat shock protein 47 (Hsp47) and their implications for collagen biosynthesis.
Heat shock protein 47 (Hsp47) is a procollagen/collagen-specific molecular chaperone protein derived from the serpin family of proteins and essential for the early stages of collagen biosynthesis. In this paper, the results of an extensive biophysical analysis of mature recombinant mouse Hsp47 show the existence of both a structurally mesostable monomer with a 5-strand A-sheet and/or a hyperstable trimer; both states have biological activity. It is also demonstrated that Hsp47 is able to bind to a monomeric and partially folded conformation collagen mimic peptide (PPG)(10). Upon peptide binding Hsp47 has the capacity to induce the peptide backbone to fold into a polyproline type II conformation. Induction of this conformation results in (PPG)(10) peptides associating into higher order assemblies with increased stability compared with the monomeric peptide alone. These assemblies are similar to those observed by others (Go, N., and Suezaki, Y. (1973) Biopolymers 12, 1927-1930; Engel, J., Chen, H. T., Prockop, D. J., and Klump, H. (1977) Biopolymers 16, 601-622) when the peptide is dissolved at high concentration and are proposed to be long chains of peptides in a collagen-like configuration. Examination of the biophysical characteristics of both monomeric and trimeric Hsp47-(PPG)(10) complexes is also used to determine that the peptide-binding site does not reside in strand 4 position of sheet A, as observed for other serpins (Skinner, R., Chang, W. S. W., Jin, L., Pei, X., Huntington, J. A., Abrahams, J. P., Carrell, R. W., and Lomas, D. A. (1998) J. Mol. Biol. 283, 9-14), leaving earlier proposals of a binding site near helix A viable.
Animals↗
Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation.
Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for approximately 25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.
Carcinoma, Small Cell↗
Access to the inaccessible sequence of cpn 60.1 (195-217) by temporary oxazolidine protection of selected amide bonds.
The solid-phase peptide synthesis of a reportedly inaccessible peptide sequence of chaperonin 60.1 (195-219) is described using oxazolidine containing dipeptide building blocks ('pseudo-proline' dipeptide units). Two attempts at the synthesis of the chaperonin 60.1 sequence are outlined using one and two pseudo-proline units, respectively, and these results are compared with the outcome of an ordinary stepwise (double) coupling procedure. The only successful synthesis is that combining two pseudo-proline building blocks.
Amides↗
A novel peptide, THALWHT, for the targeting of human airway epithelia.
Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.
Amino Acid Sequence↗
Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core.
Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.
Animals↗
Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34(+) cells emphasize limited transduction at low receptor levels.
Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene expression. Indirect evidence on the basis of RNA analysis and in vivo competitive repopulation experiments in animal models suggests a correlation between transduction efficiency and the abundance of retrovirus receptors on the hematopoietic target cell. However, transduction by oncoretroviral vectors is also subject to other factors such as target cell cycle status and the composition of the virus-containing medium, making it difficult to determine the level of receptor expression required for efficient transduction. In the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelope protein in a tetracycline-inducible tissue culture model that allowed for the cell cycle-independent, regulated expression of the GALV receptor (Pit1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RNA expression by 4.5-fold resulted in a mean 150-fold increase in transduction efficiency. We then analyzed cell surface expression of the Pit1 receptor using a fusion protein consisting of GALV SU portion of the viral envelope protein linked to the human IgG Fc. These experiments showed that tetracycline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding increased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells revealed increased expression of retrovirus receptors after cytokine stimulation, although overall receptor expression in CD34(+)cells remained lower than in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.
Antigens, CD34↗
The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties.
One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degrees C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.
Active Transport, Cell Nucleus↗