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Biomedical subjects

A D Miller

Publications and source records attributed to A D Miller.

At least 199 records · Page 11Linked to original sources
PubMed1990

Diaphragmatic and external intercostal muscle control during vomiting: behavior of inspiratory bulbospinal neurons.

1. The role of dorsal and ventral respiratory group (DRG and VRG) bulbospinal inspiratory (I) neurons in the control of diaphragmatic and external intercostal (inspiratory) muscle activity during vomiting was examined by recording from these neurons during fictive vomiting in decerebrate, paralyzed cats. Fictive vomiting was defined by a characteristic series of bursts of coactivation of phrenic and abdominal muscle nerves, elicited either by electrical stimulation of abdominal vagal afferents or by emetic drugs, which would be expected to produce vomiting if the animals were not paralyzed. 2. Data were recorded from 22 DRG and 29 VRG I neurons that were antidromically activated from the fourth cervical spinal segment (C4). Only 10% (5/51) of these neurons started to fire near the beginning of phrenic discharge during fictive vomiting and thus had the appropriate discharge pattern to contribute to the initial activation of the diaphragm and coactive external intercostal muscles during vomiting. The frequency of occurrence of these Active neurons was not significantly different in the DRG (3/22) and VRG (2/29) (chi 2 test). Most remaining neurons were either totally silent (n = 7) or had only sporadic, infrequent firing (n = 16) (Silent neurons, 23/51 = 45%), or else fired near the end of phrenic discharge during fictive vomiting (End neurons, 21/51 = 41%). Two neurons were categorized as having miscellaneous (Misc) behavior. 3. No differences were found among neurons having different response patterns during fictive vomiting in regard to the following: the manner in which fictive vomiting was elicited: cell location: conduction velocity; and neuronal firing onset, rate, and pattern during respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase).

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.

Amino Acid Sequence↗
PubMed1989

Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines.

Adenosine deaminase (ADA) deficiency is associated with a fatal severe combined immunodeficiency. Because most patients do not have a suitable marrow donor, the introduction of a normal ADA gene into the patient's marrow cells is a potentially useful alternative therapy. To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviral vectors containing the ADA gene under the transcriptional control of the promoter/enhancers of Moloney murine leukemia virus, the simian virus 40 early region, the cytomegalovirus immediate-early gene, the lymphotropic papovavirus, and the human beta-globin gene. ADA expression from these vectors was monitored in the ADA- human histiocytic lymphoma cell line DHL-9, and in the multipotential chronic myeloid leukemia cell line K562. ADA expression in infected K562 cells was also measured after induction of megakaryoblastic differentiation by phorbol ester, and after induction of erythroid differentiation by sodium n-butyrate or hemin. In these hematopoietic cell lines, the vectors that contained ADA controlled by either the Moloney murine leukemia virus promoter (LASN) or the cytomegalovirus promoter (LNCA) expressed ADA at much higher levels than the other vectors tested. Furthermore, in K562 cells infected with LASN and LNCA vectors, induction of terminal differentiation resulted in the same or higher level expression of ADA. These cell lines have permitted the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that provide a model for bone marrow-targeted gene therapy.

Adenosine Deaminase↗
PubMed1989

Brainstem projections to cats' upper lumbar spinal cord: implications for abdominal muscle control.

Unilateral L1-L2 gray matter HRP injections labeled neurons bilaterally in nucleus retroambiguus (expiratory neuron region of caudal ventral respiratory group) and ventromedial reticular formation. Minor labeling occurred in raphe, vestibular, and medial parabrachial nuclei, lateral reticular formation, and C1-C2. Midsagittal lesions between C1 and obex prevented nucleus retroambiguus labeling (except 1 contralateral cell adjacent an incomplete lesion), indicating that these neurons decussate between C1 and obex and have collaterals that recross the spinal cord.

Abdominal Muscles↗
PubMed1989

Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase (p56lck).

The protein tyrosine kinase p56lck is implicated in the control of lymphocyte growth by virtue of its overexpression in some lymphoid malignancies and its transforming activity in heterologous systems. Previous studies have demonstrated that levels of lck mRNA and of p56lck decline rapidly after T cell activation. The disappearance of p56lck results primarily from post-translational conversion of p56lck to more slowly migrating forms with apparent sizes of approximately 60 kDa. This modification can be provoked by treatment of lymphocytes with PMA, and has been associated with increased serine phosphorylation of the p56lck molecule. Here we demonstrate that conversion of p56lck to p60lck is a feature of the physiologic activation of T lymphocytes by antigen-presenting cells. In addition, we show that the PMA-induced modification of p56lck proceeds via a mechanism distinct from conventional protein kinase C activation. The rapid conversion of p56lck to p60lck after antigenic stimulation is consistent with the view that this membrane-associated protein tyrosine kinase regulates some aspects of the lymphocyte activation sequence.

Adult↗
PubMed1989

Long-term expression of the human beta-globin gene after retroviral transfer into pluripotent hematopoietic stem cells of the mouse.

We have studied the regulation of the human beta-globin gene after retroviral transfer into a variety of transformed and normal hematopoietic cells. After transfer into murine erythroleukemia cells (MEL) expression from the human beta-globin gene responds to inducers of erythroid maturation in parallel to the endogenous murine globin genes. After infection of human BFU-E, RNA expression from the virally-transferred beta-globin gene was measured at 2.5%-5% of the endogenous beta-globin level. The most improved globin vectors can transfer the human beta-globin gene into pluripotent hematopoietic stem cells in mouse bone marrow. Mice reconstituted with infected marrow show human beta-globin RNA and protein expression in peripheral blood cells for over 4 months. In these animals, both myeloid and lymphoid cells carry the integrated provirus at a level of about 1 copy per cell. In serial transplantation experiments, bone marrow from these animals is capable of repopulating secondary and tertiary recipient animals which go on to show long-term human beta-globin expression. Retroviral vectors thus provide a practical way to refine models of globin gene regulation through in vivo tests and to evaluate the feasibility of protocols for gene addition therapy.

Animals↗
PubMed1989

Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Adipose Tissue↗
PubMed1989

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

Animals↗
PubMed1989

Regulated expression of the human beta-globin gene after retroviral transfer into murine and human hematopoietic cells.

Retroviral vectors and infection protocols were developed which permit transfer in vitro of the human beta-globin gene into transformed erythroid cells and normal human and murine hematopoietic cells. In murine erythroleukemia (MEL) cells, RNA expression from the human beta-globin gene was regulated in parallel with the endogenous globin genes and this RNA directed synthesis of human beta-globin protein chains. Human BFU-E which were present in normal bone marrow samples were also infected with the globin virus. After erythroid maturation in vitro, several percent of the total beta-globin mRNA was derived from the virally transferred beta-globin gene in the erythroid progeny cells of the bursts. The initial design of the beta-globin vectors was improved after the removal of sequences which interfered with the production of high-titer retrovirus stocks. The improved vector can transfer the human beta-globin gene to pluripotential hematopoietic stem cells (PHSC) of the mouse as shown by long-term expression of human beta-globin RNA and protein in peripheral blood, and the presence of the globin provirus in reconstituted myeloid and lymphoid cell lineages in primary and secondary recipients of virus-infected bone marrow.

Animals↗
PubMed1989

Improved retroviral vectors for gene transfer and expression.

We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.

Animals↗
PubMed1989

Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture.

Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU-GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

Animals↗
PubMed1989

Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B.

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Animals↗
PubMed1988

Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound to the protein through the sulphur atom of cysteine-242.

The pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli is bound to the protein through the sulphur atom of a cysteine residue [Hart, Miller & Battersby (1988) Biochem. J. 252, 909-912; Beifuss, Hart, Miller & Battersby (1988) Tetrahedron Lett. 29, 2591-2594]. We show that the pyrromethane-binding residue is cysteine-242.

Amino Acid Sequence↗
PubMed1988

Central motor program for relaxation of periesophageal diaphragm during the expulsive phase of vomiting.

Relaxation of the periesophageal diaphragm during expulsion in decerebrate cats was found to arise from central mechanisms rather than a reflex of peripheral origin. Reductions in periesophageal EMG activity during expulsion were similar before and after cervical vagotomy, which abolishes reflex relaxation of the periesophageal diaphragm following esophageal distension. Reduced activity was also observed in a phrenic nerve branch to the periesophageal region during 'fictive vomiting' in a paralyzed animal.

Animals↗
PubMed1988

Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound through the sulphur atom of a cysteine residue.

Hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli uses a novel pyrromethane cofactor to bind the growing pyrrolic chain for hydroxymethylbilane biosynthesis [Hart, Miller, Leeper & Battersby (1987) J. Chem. Soc. Chem. Commun. 1762-1765]. We show that this cofactor is bound to the protein through the sulphur atom of a cysteine residue.

Ammonia-Lyases↗
PubMed1988

Retroviral transfer of genes into canine hematopoietic progenitor cells.

Amphotropic retroviral vectors containing either the bacterial neomycin phosphotransferase gene or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. Successful transfer and expression of both genes in canine hematopoietic progenitor cells has been achieved as measured by the ability of the viruses to confer resistance to either methotrexate (MTX) or the aminoglycoside G418, respectively. Gene transfer was achieved using helper-free retroviral vectors. The rate of gene expression in canine granulocyte/macrophage colony-forming units (CFU-GM) after cocultivation for 24 hours with virus-producing packaging cells ranged from 6-25%. Autologous marrow cocultivated for 24 hours with virus-producing packaging cells was transplanted into six dogs after lethal total body irradiation. All dogs showed engraftment within two weeks and four dogs survived for 5-7 months without adverse effects. One dog that had been given marrow infected with a DHFR* virus and that received MTX as in vivo selection after marrow transplantation and survived, showed 0.1 and 0.03% MTX-resistant CFU-GM at weeks 3 and 5. The efficiency of gene transfer into canine CFU-GM has been increased threefold by culturing marrow cells for six days in long-term marrow culture after 24 hour cocultivation with virus producing packaging cells.

Animals↗