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Biomedical subjects

A D Miller

Publications and source records attributed to A D Miller.

At least 145 records · Page 8Linked to original sources
PubMed1994

A family of retroviruses that utilize related phosphate transporters for cell entry.

The amphotropic murine retrovirus receptor Ram-1 shows significant sequence similarity to the gibbon ape leukemia virus (GALV) receptor Glvr-1, and both of these cell surface virus receptors normally function as sodium-dependent phosphate symporters. However, Ram-1 from humans or rats does not serve as a receptor for GALV, and Glvr-1 from humans does not serve as a receptor for amphotropic virus. Here we show that the murine retrovirus 10A1 can enter cells by using either Glvr-1 or Ram-1. Furthermore, we have constructed Ram-1/Glvr-1 hybrid receptors that allow entry of both GALV and amphotropic virus. While GALV and amphotropic virus are in separate interference groups when assayed on human cells, they do interfere with each other in cells expressing the hybrid receptor. These results indicate a close functional relationship between retroviruses that utilize members of this newly defined receptor family and provide a molecular explanation for nonreciprocal and cell type-specific interference observed for some retrovirus classes.

Amino Acid Sequence↗
PubMed1994

DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors.

None of the vector systems currently available for gene therapy applications have been shown to be capable of both efficient gene transfer into nondividing cells and long-term expression through stable integration into host cell DNA. While integrating vectors based on adeno-associated virus are capable of mediating gene transfer into nondividing cells, this process is 200-fold less efficient than transduction of dividing cells. We demonstrate that the transduction efficiency of adeno-associated virus vectors can be increased by treatment with DNA-damaging agents. Nondividing cells are especially responsive, with increases in transduction efficiency of up to 750-fold. This finding has the potential to facilitate gene therapy applications requiring gene transfer to nondividing cells.

Alkaline Phosphatase↗
PubMed1994

Organization of vestibular inputs to nucleus tractus solitarius and adjacent structures in cat brain stem.

The vestibular system is involved in maintaining stable blood pressure and respiration during changes in posture and is essential for eliciting motion sickness-related vomiting. Because the nucleus tractus solitarius (NTS) participates in the regulation of sympathetic and inspiratory outflow and the triggering of emesis, we tested the hypothesis that this region receives vestibular inputs in cats. In one set of experiments, microinjections of the tracer Phaseolus vulgaris leucoagglutinin into the medial and inferior vestibular nuclei labeled projections to the middle and lateral regions of the NTS. In electrophysiological experiments, electrical stimulation of the vestibular nerve modified the firing rates of neurons located in the same regions. Some neurons with vestibular inputs received convergent signals from the abdominal vagus nerve and could potentially mediate motion sickness-related vomiting. Others received convergent baroreceptor inputs and could act as a substrate for some components of vestibulosympathetic reflexes. In contrast, inspiratory neurons in the dorsal respiratory group received little vestibular input, suggesting that vestibulorespiratory reflexes are mediated by cells located elsewhere.

Animals↗
PubMed1994

Properties of sympathetic reflexes elicited by natural vestibular stimulation: implications for cardiovascular control.

1. To study the properties of vestibulosympathetic reflexes we recorded outflow from the splanchnic nerve during natural vestibular stimulation in multiple vertical planes in decerebrate cats. Most of the animals were cerebellectomized, although some responses were recorded in cerebellum-intact preparations. Vestibular stimulation was produced by rotating the head in animals whose upper cervical dorsal roots were transected to remove inputs from neck receptors; a baroreceptor denervation and vagotomy were also performed to remove visceral inputs. 2. The plane of head rotation that produced maximal modulation of splanchnic nerve activity (response vector orientation) was measured at 0.2-0.5 Hz. The dynamics of the response were then studied with sinusoidal (0.05- to 1-Hz) stimuli aligned with this orientation. 3. Typically, maximal modulation of splanchnic nerve outflow was elicited by head rotations in a plane near pitch; nose-up rotations produced increased outflow and nose-down rotations reduced nerve discharges. The gains of the responses remained relatively constant across stimulus frequencies and the phases were consistently near stimulus position, like regularly firing otolith afferents. Similar response dynamics were recorded in cerebellectomized and cerebellum-intact animals. 4. The splanchnic nerve responses to head rotation could be abolished by microinjections of the excitotoxin kainic acid into the medial and inferior vestibular nuclei, which is concordant with the responses resulting from activation of vestibular receptors. 5. The properties fo vestibulosympathetic reflexes recorded from the splanchnic nerve support the hypothesis that the vestibular system participates in compensating for posturally related changes in blood pressure.

Animals↗
PubMed1994

Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.

Adenosine Deaminase↗
PubMed1994

Effects of immediate and delayed error correction on the acquisition and maintenance of sight words by students with developmental disabilities.

We compared immediate and delayed error correction during sight-word instruction with 5 students with developmental disabilities. Whole-word error correction immediately followed each error for words in the immediate condition. In the delayed condition, whole-word error correction was provided at the end of each session's three practice rounds. Immediate error correction was superior on each of the four dependent variables.

Behavior Therapy↗
PubMed1994

Emetic reflex arc revealed by expression of the immediate-early gene c-fos in the cat.

The organization of the central neuronal circuitry that produces vomiting was explored by mapping the distribution of c-fos protein (Fos)-like immunoreactivity (FLI) as a monitor of functional activity. The brainstem and spinal cord were examined in cats administered multiple emetic drugs (cisplatin, lobeline, protoveratrine, naloxone, apomorphine) or control saline injections. Some animals were decerebrated, paralyzed, and artificially ventilated to avoid possible Fos expression induced by sensory feedback or fluid depletion during vomiting. Fictive vomiting was identified in these animals by a characteristic pattern of respiratory muscle nerve (phrenic and abdominal) coactivation. Tissues were immunoprocessed using an antibody raised against amino acids 1-131 of Fos and the avidin-biotin peroxidase complex method. Enhanced nuclear FLI was observed in experimental animals along portions of the sensorimotor emetic reflex arc, including the nodose ganglia, area postrema, nuclei of the solitary tract (especially medial and subpostrema subnuclei), intermediate reticular zone of the lateral tegmental field, nucleus retroambiguus, C2 inspiratory propriospinal cell region, and dorsal vagal and phrenic motor nuclei. Enhanced FLI was also detected in the raphe magnus, subretrofacial nucleus, and spinal dorsal horn. Regions showing no recognizable differences in FLI between experimental and control animals included the vestibular, cochlear, spinal trigeminal, subtrigeminal, and lateral reticular nuclei. Only minor differences were observed in the distributions of FLI between intact and decerebrate animals. No unique, well-defined group of labeled neurons that might function as a "vomiting center" could be identified. Instead, the pattern of c-fos expression suggests that neurons involved in coordinating the emetic response may radiate from the area postrema and nucleus of the solitary tract to an arc in the lateral tegmental field implicated in somato-autonomic integration.

Animals↗
PubMed1993

Vestibular effects on respiratory outflow in the decerebrate cat.

Recordings were made from phrenic, abdominal and intercostal nerves following electrical stimulation of the vestibular nerve to test for the presence of vestibulo-respiratory connections in cats that were decerebrate, paralyzed, and artificially ventilated. Short stimulus trains (2 or 3 shocks) at intensities < or = 125 microA typically elicited responses bilaterally in all of the respiratory nerves; the onset latency of the evoked activity was < 15 ms from the effective shock. The mean peak-to-peak amplitudes of integrated vestibulo-respiratory responses were 15% of the average amplitude of spontaneous respiratory-related discharges in the case of the phrenic nerve and 100% in the case of the abdominal nerve. The vestibulo-respiratory reflexes, as well as vestibulo-sympathetic responses recorded from the splanchnic nerve, could be abolished by injections of the excitotoxin kainic acid confined primarily to the medial and adjacent inferior vestibular nuclei. The physiological role of vestibulo-respiratory connections is yet to be determined, but possible functions include adjustments of respiration during changes in posture, assistance in venous return to the heart during movements that might lead to orthostatic hypotension, and direct participation in the execution of specific movements and the maintenance of some postures.

Animals↗
PubMed1993

Analysis of dystrophin expression after activation of myogenesis in amniocytes, chorionic-villus cells, and fibroblasts. A new method for diagnosing Duchenne's muscular dystrophy.

BACKGROUND: DNA analysis of peripheral-blood leukocytes is routinely used to demonstrate mutations in the dystrophin gene in patients with Duchenne's or Becker's muscular dystrophy. In approximately 35 percent of patients, DNA studies are not informative; in these patients immunochemical analysis of a muscle-biopsy specimen can determine whether dystrophin, the protein product of the gene for Duchenne's dystrophy, is present at reduced levels or absent. DNA analysis can be performed in amniocytes or chorionic-villus cells to identify mutations of the dystrophic gene prenatally, but immunochemical testing for dystrophin cannot be performed because the protein is not expressed in these cells. METHODS: To circumvent this limitation in prenatal diagnosis, we induced myogenesis in 21 cultures of skin fibroblasts, 49 amniocyte cultures, and 6 chorionic-villus cell cultures by infecting the cells with a retrovirus vector containing MyoD, a gene regulating myogenesis. Transfection of MyoD into cells that do not normally develop into muscle cells results in the production of a protein that switches on myogenesis. We performed immunocytochemical analysis for dystrophin in the MyoD-converted muscle cells. RESULTS: We found that 60 of 61 myotube cultures from subjects with no family history of Duchenne's dystrophy expressed dystrophin. Both myotube cultures from the two patients with Becker's dystrophy also expressed dystrophin, but all cultures from nine patients and two fetuses with Duchenne's dystrophy were dystrophin-deficient. CONCLUSIONS: Immunocytochemical analysis of dystrophin in genetically altered non-muscle cells is feasible and may be applicable to the prenatal and postnatal diagnosis of Duchenne's muscular dystrophy when conventional DNA analysis is not informative.

Amniotic Fluid↗
PubMed1993

Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter.

In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.

Animals↗
PubMed1993

Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES) do not catalyse the refolding of mitochondrial malate dehydrogenase.

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.

Adenosine Triphosphate↗
PubMed1993

Evaluation of role of upper cervical inspiratory neurons in respiration, emesis and cough.

Upper cervical (C1-3) inspiratory (UCI) propriospinal neurons project to spinal segments containing respiratory motoneurons, but their functional significance is unknown. Bilateral kainic acid injections into this cell column in 12 decerebrate cats (11 paralyzed and artificially ventilated) had no major effect on phrenic, intercostal, and abdominal nerve discharge or EMG activity during (fictive) respiration, vomiting and coughing. Thus, UCI neurons are unessential for activation of major respiratory muscles during these behaviors.

Abdomen↗
PubMed1993

Respiratory interneurons of the lower cervical (C4-C5) cord: membrane potential changes during fictive coughing, vomiting, and swallowing in the decerebrate cat.

The possible roles of interneurons in the C4-C5 cervical spinal cord in conveying central drives to phrenic motoneurons during different behaviour patterns were investigated using intracellular recordings in decerebrate, paralysed, artificially ventilated cats. Eleven cells were tentatively classified as respiratory interneurons since they: (i) could not be antidromically activated from the ipsilateral whole intrathoracic phrenic nerve, and (ii) exhibited large membrane potential changes during eupnea (7.3 mV +/- 3.6, range 2-13.5 mV) or non-respiratory behaviour patterns. Six neurons depolarized in phase with phrenic discharge; four others depolarized during the expiratory phase; one neuron exhibited depolarization during the end of both expiration and inspiration. A variety of responses was observed during fictive coughing, vomiting, and swallowing. The results are consistent with C4-C5 expiratory interneurons conveying inhibition to phrenic motoneurons during different behaviour patterns. The responses of inspiratory and multiphasic neurons suggest that the roles of these interneurons are mode complex than simply relaying central excitatory or inhibitory drive to phrenic motoneurons.

Animals↗
PubMed1993

Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production.

A variety of retroviral vectors for transduction and expression of clotting factor VIII (FVIII) were constructed by using truncated forms of a FVIII cDNA lacking part or all of the nonessential B-domain sequences. Both the titer of virus and FVIII protein production from the vectors was about 2 orders of magnitude lower than the virus titer and protein production from identical retroviral vectors containing other cDNAs, including clotting factor IX. These decreases could be entirely explained by an observed 100-fold lower accumulation of vector RNAs containing the FVIII sequences in comparison to vectors containing other cDNA sequences. Deletion analysis of one of the FVIII vectors demonstrated that diffuse sequences within the FVIII coding region had a deleterious effect upon vector titer and RNA accumulation. One inhibitory signal could be localized to a 1.2-kb stretch of DNA, but further localization was not possible as additional size reduction abolished the activity. These results indicate that expression of FVIII is regulated by signals within FVIII coding sequence that result in decreased RNA accumulation and FVIII protein production. Alteration of these inhibitory signals to permit high-level FVIII production may be difficult due to the wide distribution of these signals within the coding region of the protein.

3T3 Cells↗
PubMed1993

The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4.

The nef gene product of human immunodeficiency virus type 1 has been shown to induce CD4 downregulation from the surface of human cells. To determine if this effect is species specific, we used a retroviral vector to transduce the human immunodeficiency virus type 1 nef gene into murine cells expressing human, chimpanzee, or murine CD4. Our results indicate that Nef induces cell surface downregulation of all three molecules. We also determined that Nef is functional in murine T cells and induces downregulation of both murine CD4 and CD8 (Ly-2) from the cell surface. In contrast, Nef does not downregulate cell surface expression of human CD8 in either murine or human cells. By using a mutant of human CD4 lacking its cytoplasmic domain and a human CD4/CD8 chimera, we determined that the cytoplasmic domain of CD4 is required for its downregulation by Nef. Transduction with a control vector had no effect on CD4 cell surface levels, indicating that retroviral transduction by itself has no significant effect on the cell surface levels of CD4. These results show that the observed downregulation of CD4 by Nef is independent of human-specific factors, is not species specific, and requires the cytoplasmic domain of CD4.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera.

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.

3T3 Cells↗
PubMed1992

Human gene therapy comes of age.

Advances in the understanding of molecular biology of human disease and the development of efficient gene transfer techniques have resulted in practical approaches to human gene therapy, with new techniques being developed at an increasing rate. The first trials have now begun in humans and initial results are positive.

Genetic Diseases, Inborn↗