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Biomedical subjects

D Young

Publications and source records attributed to D Young.

At least 253 records · Page 14Linked to original sources
PubMed1996

Peptidergic regulation of chromatophore function in the European cuttlefish Sepia officinalis

Color patterning in cephalopod molluscs involves activation of a peripheral chromatophore system that is under neuromuscular control. The complex behavior of individual chromatophores is mediated by a specific set of muscles, the chromatophore muscles, that receive direct innervation from the central nervous system. To date, glutamate is the only excitatory transmitter that has been proposed to act at the chromatophore neuromuscular junction of cephalopods. We present data demonstrating that the chromatophore muscles in the European cuttlefish Sepia officinalis are also regulated by the FMRFamide family of neuropeptides. Using an in vitro chromatophore bioassay, it has been determined that several FMRFamide-related peptides (FaRPs) are potent excitors of the chromatophore muscles, causing chromatophore expansion. Immunocytochemical analyses of the central nervous system using an FMRFamide antibody revealed the presence of FMRFamide-like immunoreactive cell bodies in the posterior chromatophore lobes, the region of the brain containing the chromatophore motoneurons of the fin and mantle. FMRFamide-like immunoreactivity was also seen in the periphery, in the nerves around the chromatophores and in close apposition to the muscles in the chromatophore layer of the fin. HPLC analysis of the fin dermis isolated four bioactive peaks that were FMRFamide-immunoreactive when tested on an immunoblot assay. Two of these peaks co-eluted with known FaRPs, FMRFamide and ALSGDAFLRFamide, a decapeptide isolated from squid. Taken together, these data suggest that the FaRPs are likely to be endogenous excitors of the chromatophore muscles in cephalopods.

Journal Article↗
PubMed1996

Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells.

As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34+ cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34+ cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34+ cells into cycle were stem cell factor (SCF) plus one of the following: interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF). The maximum numbers of cells in S+G2M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34+ cells remained quiescent in the first G0/G1 phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34+CD38- subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-G pseudotyped virus containing the neomycin phospho-transferase gene (neo), we exposed CD34+ cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34+ cells and the CD34+CD38- subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT-neoDNA was detectable in CD34+ cells that have been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34+ cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the 34+CD38- subset showed that RT-reoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34+ cells but not in CD34+CD38- cells. These results suggest during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34+CD38-cells in addition to the lack of mitotic activity.

ADP-ribosyl Cyclase↗
PubMed1996

Bone marrow engraftment following unrelated donor transplantation utilizing busulfan and cyclophosphamide preparatory chemotherapy.

Two major problems of unrelated donor transplantation have been an increased incidence of GVHD and graft failure. Even with HLA identity by microlymphocytotoxicity assay and non-reactive MLC, URD marrow transplant recipients have a higher incidence of graft rejection and GVHD. The preparative regimen busulfan 16 mg/kg and cyclophosphamide 120 mg/kg (BuCy2) has been shown to be at least as effective in preparation of recipients with CML of HLA-identical sibling grafts as cyclophosphamide and total body irradiation (Cy/TBI). However, concern about a high rejection rate in URD transplants has prevented most centers from using BuCy2 in this setting. From March 1990 to March 1994, 26 patients underwent URD transplantation following preparation with BuCy2. Patients received either standard cyclosporine and methotrexate or cyclosporine and methylprednisolone for GVHD prophylaxis. Two patients died on day 16 and 20 without evidence of hematopoietic engraftment. Of the 24 patients evaluable for engraftment, 23 (96%) had evidence of donor engraftment defined as an ANC > 0.5 x 10(9)/1. No patient who had initial engraftment had late graft failure. Within our study group the risk of graft rejection or graft failure does not appear to be higher than that reported for URD transplants utilizing TBI-containing regimens.

Acute Disease↗
PubMed1996

A safety and efficacy comparison study of two dosing regimens of epoetin alfa in patients undergoing major orthopedic surgery.

Previous studies have demonstrated the efficacy of perioperative Epoetin alfa in decreasing allogeneic transfusion exposure in patients undergoing orthopedic surgery. A randomized, multicenter trial was conducted comparing the safety and efficacy of a weekly Epoetin alfa dosing regimen in patients with hemoglobin levels > or = 10 to < or = 13 g/dL scheduled to undergo major elective orthopedic arthroplasty, with a daily regimen previously shown to be effective. patients on the weekly regimen showed a greater baseline-to-presurgery hemoglobin increase versus the daily regimen group (1.44 +/- 1.029 g/dL versus 0.73 +/- 0.867 g/dL). Moreover, the weekly 600 IU/kg regimen was similar to the daily 300 IU/kg regimen with respect to safety and the avoidance of allogeneic transfusion. These data showed the weekly Epoetin alfa regimen to be at least as efficacious as the daily regimen and more convenient.

Aged↗
PubMed1996

Pluripotent and lineage-committed CD34+ subsets in leukapheresis products mobilized by G-CSF, GM-CSF vs. a combination of both.

To define an optimal regimen for mobilizing blood-derived progenitor cells from healthy donors for allogeneic transplantation, we have studied the early and lineage-committed CD34+ subsets in the leukapheresis products after mobilization with G-CSF (10 micrograms/kg/d), GM-CSF (10 micrograms/kg/d), and the combination of G-CSF and GM-CSF (G/GM, 5 micrograms/kg/d of each). We used three color and five dimensional flow cytometry with a panel of monoclonal antibodies against CD3, CD7, CD10, CD11b, CD15, CD33, CD34, CD38, CD45, CD61, and CD71. As reference, we also analyzed CD34+ subsets in samples from umbilical cord blood (UCB) and from adult bone marrow (BM). The level of total CD34+ cells was 0.04 +/- 0.03% (mean +/- SD) in peripheral blood at baseline, and reached a maximum on day 5 or day 6 of administration of growth factors. The percentages of CD34+ cells in the leukapheresis products were 1.06 +/- 0.37% (mean +/- SD) with G-CSF mobilization, 0.35 +/- 0.24% with GM-CSF, and 0.92 +/- 0.61% with the combination of both. Among the CD34+ subsets, the percentage of cells that were CD34+/CD38- was highest in UCB (7.18 +/- 5.58%) and lowest in G-CSF mobilized peripheral blood (0.80 +/- 0.22%), whereas GM-CSF or G/GM mobilized products gave rise to intermediate levels (4.43 +/- 3.40%, 3.61 +/- 2.42%, respectively). The differences between G/GM and G-CSF, between UCB and G-CSF, or between UCB and BM are significant. The absolute numbers of CD34+/CD38- and CD34+/CD38-/HLA-DR+ subsets are also significantly higher in the G/GM mobilized products than in G-CSF products. The cloning efficiency of G/GM mobilized CD34+ cells was 2 times higher than that of G-CSF mobilized CD34+ cells, albeit the difference was statistically marginal. The profile of CD34+ subsets mobilized by the combination of G/GM approaches that found in UCB. Our data illustrate that different growth factors and regimens can preferentially mobilize different CD34+ subsets from normal donors, and that the combination of G-CSF and GM-CSF might be an optimal regimen.

Antigens, CD34↗
PubMed1996

Evaluation of anti-viral filters.

The purification processes used to prepare medicines from human and animal cell lines provide one of the major opportunities for virus risk reduction. The purification process can be validated for its ability to remove a range of viruses and can be shown to provide a significant barrier to the transmission of viruses from cell substrates and raw materials to the patient. Ultrafiltration membranes which are manufactured with extremely tight control on pore size distribution can remove a broad spectrum of viruses with high efficiency. A scheme is described for the evaluation of anti-viral filters and the results from evaluation of two filters are presented. The experiments carried out with ultrafiltration membranes suggest that they can provide significant additional virus risk reduction factors for biopharmaceuticals.

Animals↗
PubMed1995

Differential expression of inducible transcription factors in basal ganglia neurons.

The dopamine receptor antagonist, haloperidol, produced a time-dependent differential induction of inducible transcription factors (ITFs) in rat striatal neurons: Fos, Fos B, Jun B, Jun D, Krox 20, and Krox 24, but not c-Jun, were induced in the caudate putamen and nucleus accumbens with varying time courses. The induction of Fos by haloperidol was stronger in anterior versus posterior regions of the striatum. In contrast, induction of Fos by the muscarinic agonist pilocarpine was stronger in the posterior regions of the striatum suggesting that muscarinic receptors do not play a role in the induction of ITFs in striatal neurons by haloperidol. Although c-Jun was not induced in caudate neurons by haloperidol it was strongly induced in these neurons following prolonged seizure activity. The differential pattern of Jun protein expression suggests that haloperidol induces a specific transcriptional program in basal ganglia neurons. These effects of haloperidol may be involved in producing its extrapyramidal side effects.

Animals↗
PubMed1995

Differential expression of CAP and CAP2 in adult rat tissues.

We previously reported the identification of the human CAP and CAP2 genes which encode proteins related to the yeast adenylyl cyclase (CYR)-associated CAP protein. The rat CAP homolog, MCH1, has also been previously cloned. We have cloned a cDNA encoding the rat homolog of CAP2. Rat CAP/MCH1 and CAP2 are 63% identical to each other. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we have examined CAP/MCH1 and CAP2 mRNA levels in various adult rat tissues. Our results show a dramatic difference in the pattern of expression of these two genes. Consistent with previous reports, we detected CAP/MCH1 mRNA in all tissues examined; however, levels vary substantially between tissues. In particular, we found that CAP/MCH1 mRNA are present at relatively high levels in spleen, testes and lung, at moderate levels in brain, kidney, liver and small intestine, and at significantly lower levels in heart, skeletal muscle and skin. We have also investigated the levels of CAP/MCH1 in rat tissues by immunoblotting with a polyclonal antibody raised against a human CAP::GST fusion protein. In general, we find that the CAP/MCH1 mRNA levels reflect the amount of CAP/MCH1 found in different tissues. In contrast, CAP2 transcripts were present at relatively high levels in testes, at moderate levels in brain, heart and skeletal muscle, at lower levels in lung, skin, kidney and small intestine, and were undetectable in liver or spleen. The differences between the sequences and expression patterns of CAP/MCH1 and CAP2 are significant and suggest that these proteins have distinct functional roles.

Adaptor Proteins, Signal Transducing↗
PubMed1995

X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions.

The X-ray structure of the tetrameric iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been refined to an R-factor of 0.167 and a correlation coefficient of 0.954 at 2.0 A resolution. The crystals are monoclinic P2(1) and have four subunits related by strong non-crystallographic 222 (or D2) symmetry in the asymmetric unit. 198 of the 207 amino acids of each subunit are defined by the electron density which shows that they adopt the conserved fold of other iron- or manganese-dependent SODs. The structure can be divided into two domains, the N-terminal domain involving an extended region followed by two projecting antiparallel alpha-helices, and the C-terminal domain containing four more helical segments with a three-stranded antiparallel beta-sheet inserted sequentially between the fourth and fifth helices. The catalytic iron is co-ordinated by five ligands: three histidines (residues 28, 76 and 164), one aspartate (160) and a solvent molecule. The inferred positions of protons at the active site are consistent with the solvent ligand being a hydroxide ion. This ligand interacts with His145 in the Mycobacterium tuberculosis SOD. In the highly homologous Mycobacterium leprae Mn-SOD, the histidine is replaced by glutamine, this being the only significant residue difference within 10 A of the Fe3+. The nature of the amino acid at this position may influence the metal ion specificity of these enzymes. The subunits of the Mycobacterium tuberculosis SOD associate by polar contacts to form dimers, which closely resemble those of other dimeric or tetrameric Fe- or Mn-SODs. However, the dimer-dimer interactions within the tetramer are novel, being dominated by dimerisation of the 144 to 152 loop regions which connect the outer two beta-strands of the three-membered beta-sheet. This contrasts strongly with the other tetrameric Fe- or Mn-SODs where the dimer-dimer association is dominated by the projecting alpha alpha-turn in the N-terminal domain.

Amino Acid Sequence↗
PubMed1995

Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1.

Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

Antigens, CD↗
PubMed1995

Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo.

Glial-cell-line-derived neurotrophic factor (GDNF), a recently cloned new member of the transforming growth factor-beta superfamily, promotes survival of cultured fetal mesencephalic dopamine neurons and is expressed in the developing striatum. There have, however, been no reports about effects of GDNF in situ. We have used the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces parkinsonian symptoms in man, to determine whether GDNF might exert protective or regenerative effects in vivo in the adult nigrostriatal dopamine system in C57/B1 mice. GDNF injected over the substantia nigra or in striatum before MPTP potently protects the dopamine system, as shown by numbers of mesencephalic dopamine nerve cell bodies, dopamine nerve terminal densities and dopamine levels. When GDNF is given after MPTP, dopamine levels and fibre densities are significantly restored. In both cases, motor behaviour is increased above normal levels. We conclude that intracerebral GDNF administration exerts both protective and reparative effects on the nigrostriatal dopamine system, which may have implications for the development of new treatment strategies for Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗