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Biomedical subjects

D M Stone

Publications and source records attributed to D M Stone.

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

Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.).

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHSV), the rhabdovirus which causes viral haemorrhagic septicaemia (VHS) in farmed salmonid fish. Phylogenetic analysis of a 360 nt region of the glycoprotein gene from a range of marine and fresh water VHSV isolates identified three genogroups, I-III. Significantly, two virus isolates recovered from ulcerated North Sea cod caught off the Shetland Islands, and an isolate recovered from diseased turbot farmed on the island of Gigha, Scotland were assigned to the same genogroup. Moreover, a virus isolated from diseased turbot farmed on the Baltic Sea coast shared 99.4% nucleotide sequence similarity with a virus associated with a VHS outbreak in rainbow trout. This is the first time that a genetic link between a VHS outbreak and natural VHSV infections of marine fish species has been demonstrated.

Amino Acid Sequence↗
PubMed1996

The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog.

The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.

Amino Acid Sequence↗
PubMed1996

Characterization of a multicomponent receptor for GDNF.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.

Amino Acid Sequence↗
PubMed1996

Comparison of the antiviral efficacy of ribozymes and antisense RNA directed against bovine leukemia virus rex/tax.

Despite the theoretical attraction of ribozymes, which cleave their target RNA, as compared with antisense RNA, which only blocks translation, few studies have assessed the relative efficacy of ribozymes and antisense RNA directed against the identical target sequence. Previously, we described the efficacy of a hammerhead ribozyme targeted against rex/tax, a regulatory gene of bovine leukemia virus (BLV). In this study, we asked whether antisense RNA targeted against the same site would also be efficacious. BLV-infected bat lung cells were transfected with an antisense RNA-encoding plasmid under the control of sarcoma virus (RSV) promoter, and stable cell lines were selected. No inhibition of viral p24 expression was demonstrated in seven cell lines transfected with the antisense RNA targeted at the same site as the ribozyme or in three cell lines transfected with an antisense sequence targeted against the tax 5' initiation codon. Although in previous experiments antisense DNA oligonucleotides inhibited Tax expression in vitro, stably transfected plasmids encoding antisense RNA of the same sequence did not inhibit viral expression in BLV-infected cells in this study. These results suggest that in persistently infected cells producing high levels of BLV, the ribozyme is more effective than antisense RNA directed at rex/tax transcripts.

Animals↗
PubMed1996

Elevated pim-1 and c-myc proto-oncogene induction in B lymphocytes from BLV-infected cows with persistent B lymphocytosis.

Bovine leukemia virus (BLV) induces a non-malignant, polyclonal, persistent lymphocytosis (PL) of circulating, CD5 B lymphocytes in cattle, with variable progression to CD5 B cell leukemia or lymphoma. We analyzed the expression of two proto-oncogenes, pim-1 and c-myc, proto-oncogenes deregulated in some human B cell leukemias and lymphomas, in peripheral blood mononuclear leukocytes (PBML) from BLV-infected PL cows. Results demonstrate that pim-1 and c-myc mRNA levels are elevated in unfractionated stimulated PBML from a sample of PL cows naturally infected with BLV. Results confirm that pim-1 is constitutively expressed, but not inducible in normal bovine peripheral blood B lymphocytes, but can be induced in the predominantly CD5 B lymphocytes from BLV-infected PL cows. Results further demonstrate that c-myc is inducible in bovine B and T lymphocytes regardless of BLV status, but the amount of induction is greater in B lymphocytes from BLV-infected PL cows than in B lymphocytes from noninfected control cows. These results suggest that pim-1 and c-myc are upregulated in B lymphocytes from BLV-infected PL cows and that deregulation of proto-oncogene expression is not limited to completely transformed cells, but can also characterize a naturally occurring, pre-neoplastic lymphocytic state.

Animals↗
PubMed1995

Up-regulation of IL-2 receptor alpha and MHC class II expression on lymphocyte subpopulations from bovine leukemia virus infected lymphocytotic cows.

Infection with bovine leukemia virus (BLV) leads to a persistent lymphocytosis (PL) characterized by a marked increase in circulating B lymphocytes that express the orthologue of CD5. To gain insight into the factors accounting for lymphocytosis, experiments were conducted to determine the functional activation status of lymphocytes from BLV seronegative and BLV infected aleukemic cows with PL. Stimulation with the B lymphocyte mitogen Staphylococcus aureus Cowan strain I (SAC), recombinant human interleukin-2 (rIL-2), or pokeweed mitogen (PWM), a T lymphocyte-dependent B lymphocyte mitogen, revealed differences in the pattern of expression of IL-2 receptor alpha (IL-2R alpha) and major histocompatibility (MHC) class II molecules on B and T lymphocytes from uninfected and BLV infected PL cows. rIL-2 induced expression of IL-R alpha on B lymphocytes from PL cows but not B lymphocytes from BLV seronegative cows. SAC alone, or in combination with rIL-2, had no effect on B lymphocytes from BLV seronegative cows. However, rIL-2 alone or in combination with SAC induced expression of IL-2R alpha on B lymphocytes from PL cows. PWM stimulated expression of IL-2R alpha on bovine B lymphocytes regardless of BLV status, and induced a significantly higher level of expression on B lymphocytes from PL cows. Mitogens and rIL-2 had a similar stimulatory effect on induction of IL-2R alpha expression on CD4 T lymphocytes regardless of BLV status. Only PWM induced expression of IL-2R alpha on bovine CD8 T lymphocytes and induced a significantly higher level of expression on this T lymphocyte subset from PL cows. Examination of freshly isolated B lymphocytes from PL cows revealed increased spontaneous expression of the MHC class II molecule compared to B lymphocytes from control cows. None of the culture conditions examined induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV seronegative cows. In contrast, SAC+IL-2 and PWM induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV infected PL cows, resulting in a significantly greater proportions of these lymphocyte subsets expressing this molecule compared to CD4 and CD8 T lymphocytes from control cows. The data indicate that infection with BLV affects the response of B and T lymphocytes to signals of activation, up-regulating the expression of surface molecules involved in both direct contact and cytokine-mediated T lymphocyte-dependent B lymphocyte activation.

Animals↗
PubMed1995

Tissue- and age-specific expression patterns of alternatively spliced agrin mRNA transcripts in embryonic rat suggest novel developmental roles.

Agrin is an extracellular matrix protein that mediates the nerve-induced clustering of nicotinic acetylcholine receptors on target muscle cells, and thus plays a key role in development of the neuromuscular synapse. Alternative exon usage within the rat agrin gene predicts numerous protein isoforms, which differ by the inclusion or exclusion of small inserts at three sites in the C-terminal half of the molecule; the insert status at two of these sites, termed Y and Z, profoundly influences the acetylcholine receptor clustering activity. We have examined the cellular expression patterns of agrin messenger RNA transcripts during rat embryogenesis by in situ hybridization with isoform-specific probes. Six 36-mer oligonucleotide probes were designed to distinguish between mRNA isoforms at either the Y site: the encoded protein contains either no insert (Y0) or a 4-amino acid insert (Y4), or the Z site: the encoded protein contains either no insert or one of 8 (Z8), 11 (Z11), or 19 (Z19) amino acids. Strikingly different expression patterns were observed for the individual Y- and Z-site encoding messages. While optional exon usage predicts the possibility of eight different agrin isoforms at the two splice sites, we detected only four isoforms in vivo: Y4Z0, Y0Z0, Y4Z8, and Y4Z19. The Y4Z0 transcript, which comprised the majority of the agrin expressed, was localized exclusively to nervous tissue and exhibited a distribution profile suggestive of a potential role in neurogenesis and/or neural differentiation. From embryonic day 13 to birth, Y4Z0 was found in mitotic ventricular zones, spinal, cranial, and sympathetic ganglia, and diffusely throughout the brain. In contrast, Y0Z0 was not expressed in neurons, but specifically labeled capillary endothelial cells within the developing nervous system. Y4Z8 and Y4Z19, the forms most active in acetylcholine receptor aggregation, were expressed at low levels only in spinal and brainstem motor neurons; Z19 expression declined from embryonic day 15 to adulthood, whereas Z8 expression increased slightly during this period. Transcripts encoding the Z11 insert could not be detected. These data suggest potential novel biological roles for agrin beyond that originally proposed in synapse formation.

Agrin↗
PubMed1995

Olfactory marker protein mRNA is found in axons of olfactory receptor neurons.

The separation between the cell bodies of olfactory receptor neurons in the nasal cavity and their axon terminals in the olfactory bulb make them attractive for studying axonal transport. Although high molecular weight RNAs are generally believed to be excluded from axons of mature neurons, we demonstrate here that mRNA for olfactory marker protein (OMP), an abundant cytoplasmic protein selectively expressed in mature receptor cells, is present in rodent olfactory receptor axons. OMP RNA was detected by in situ hybridization at the light microscope level in axons and in terminals. By nuclease protection, the level of OMP RNA in the olfactory bulb was 5-10% of that in the olfactory epithelium where the cell bodies reside. In contrast to axonally transported vasopressin and oxytocin mRNAs, which are deficient in their 3' polyA tails, axonal OMP RNA fractionated as polyA+. OMP RNA was lost from axons and terminals after deafferentation, suggesting that OMP RNA was synthesized in receptor cell bodies in the epithelium and was transported into axons and terminals in the olfactory bulb. RNA for G(olf), a G-protein highly expressed in dendrites of mature olfactory receptor neurons, was not detected in the olfactory bulb. We hypothesize that the immature nature of the cytoskeleton and, specifically, the lack of tightly bundled microtubules allows transport of particular mRNAs in olfactory receptor axons.

Afferent Pathways↗
PubMed1994

Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method.

Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.

Animals↗
PubMed1994

Role for poliovirus protease 2A in cap independent translation.

Viral protein synthesis in poliovirus infected cells was found to be influenced by mutations in part of the viral 5'-non-coding region (NCR) in a temperature dependent manner. At elevated temperatures these mutations resulted in virus titre reductions that allowed selection of revertant viruses. Some revertants were found to have retained the 5'-NCR mutations but had compensating mutations in the 2A protease gene that were responsible for the suppression of the temperature sensitive phenotypes. The mutations in 2A enhanced viral protein synthesis at a stage when cap dependent translation was already abolished, suggesting that the virally encoded protein 2A is directly involved in the process of cap independent translation in addition to its role in abolishing cap dependent translation.

Amino Acid Sequence↗
PubMed1994

Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli.

Recombinant mouse tryptophan hydroxylase (TPH) was expressed in large quantities in Escherichia coli strain MC 1061, using a bacterial expression vector, pKS, containing the full coding region of mouse TPH. Specific polyclonal antiserum to the subunit of the recombinant mouse TPH was produced in rabbit by injecting the TPH band cut from SDS-polyacrylamide slab gels. The resultant antiserum recognized a single identical protein band (MW = 54,000) from rat dorsal raphe area, pineal gland, and brain stem by Western blot analysis. The specific activity of recombinant mouse TPH obtained was equivalent to that of TPH purified from rat brain. The recombinant mouse TPH was stable for 3 days at 4 degrees C but lost 25% of the original activity for the same period at -20 degrees C. A serotonin concentration greater than 1 mM inhibited TPH activity under our assay conditions in a concentration-dependent fashion. The recombinant mouse TPH exhibited a charge isozyme corresponding to that of pineal gland TPH as applied to chromatofocusing column chromatography. Taken together, our results show that recombinant mouse TPH, expressed in large quantities in E. coli is not only enzymatically highly active but also shares many biochemical and immunochemical properties with native TPH.

Animals↗
PubMed1994

Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment.

Tryptophan hydroxylase (TPH) is the first and presumably rate-limiting enzyme in serotonin (5-HT) biosynthesis. End-product inhibition of rate-limiting enzymes is common and 5-HT is known to inhibit TPH activity in vivo. However, it is not known whether levels of 5-HT could also be involved in the regulation of the TPH gene. In order to determine whether TPH gene regulation is dependent on the 5-HT concentration, 5-HT levels were reduced by the administration of parachlorophenylalanine (PCPA). PCPA is a potent, specific and irreversible inhibitor of TPH activity which drastically reduces 5-HT concentration in the 5-HT neurons and terminals. When PCPA was administered, TPH activity in both cell bodies and nerve terminal areas, was reduced to 10% of control values and recovered to the control levels by day 7 in raphe nucleus, and within 14 days in the hypothalamus. In serotonergic terminal areas, 5-HT could not be detected immunohistochemically at day 1, but slowly recovered within 2 weeks. At all time points examined, aromatic L-amino acid decarboxylase (AADC) levels were not changed either in the cell body or terminal areas. The steady state levels of TPH mRNA estimated by in situ hybridization increased at day 1 and returned to control levels by day 4. AADC message levels were not altered throughout the periods. These data suggest that a decrease in 5-HT concentration may lead to an up-regulation of TPH gene transcription, by an, as yet, unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The 5' noncoding region and virulence of poliovirus vaccine strains.

All three live, attenuated vaccine strains of poliovirus contain important attenuation determinants in a short conserved sequence in the 5' noncoding region. Evidence suggests these act by weakening a secondary-structural element critical for the unusual mechanism of translational initiation of picornaviruses, in which ribosomes bind directly to a site far downstream of the 5' end. Understanding the molecular basis of attenuation may allow novel vaccine strains to be designed.

Base Sequence↗
PubMed1994

Enhanced B-lymphocyte expression of IL-2R alpha associated with T lymphocytosis in BLV-infected persistently lymphocytotic cows.

Peripheral blood lymphocytes from bovine leukemia virus (BLV)-negative and BLV-infected, aleukemic cows with persistent lymphocytosis were evaluated for expression of B and T lymphocyte subset-specific molecules and co-expression of the interleukin-2 receptor alpha (IL-2R alpha) molecule. Results demonstrate enhanced mitogen-induced expression of the IL-2R alpha molecule on B lymphocytes from BLV-infected, lymphocytotic cows. Lymphocyte subset analyses further demonstrate that BLV-infected, lymphocytotic cows are not only characterized by sustained elevations in CD5+ B lymphocytes, but also show significantly elevated numbers of CD3+, CD4+, and CD8+ T lymphocytes. These results provide evidence suggesting that B lymphocytes from BLV-infected, lymphocytotic cows are more sensitive to activation signals and up-regulation of the IL-2 signaling pathway than lymphocytes from clinically normal BLV-free cows, and that T lymphocytes may be involved in the aberrant regulatory pathways underlying BLV-induced persistent B lymphocytosis.

Animals↗
PubMed1993

Adult naris closure profoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb.

Peripheral afferent innervation appears to be required for the expression of the dopamine phenotype in the rodent main olfactory bulb. Experiments utilizing neonatal naris closure as a means of sensory deprivation also suggest that odor-induced afferent activity is required for the expression of the phenotype. These experiments are confounded, however, by the significant postnatal maturation of the dopamine system. The current experiments utilized adult unilateral naris closure to address this issue. As with neonatal closure, adult deprivation produces a profound reduction in the expression of tyrosine hydroxylase (TH), the first enzyme in the dopamine biosynthetic pathway. By 4 days a small decrease is observed in TH activity and immunoreactivity. Activity reaches a nadir of 12% of control levels at about 1 month. TH mRNA is reduced similarly when analyzed at about 2 months post-closure. Glutamic acid decarboxylase protein and mRNA expression, which are co-localized with TH, remain at close to control levels indicating the continued presence of the dopamine neurons. The time-course of the loss of TH is identical to that for zinc sulphate-induced denervation of the olfactory bulb. These data support the hypothesis that odor modulated afferent activity is required for expression of the dopamine phenotype and that, if a trophic factor is involved, its release is also activity dependent.

Animals↗
PubMed1993

Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine.

In the present study, we examined time-dependent changes in activity, mRNA and immunoreactivity of the serotonin biosynthetic enzymes, tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (AADC) in dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus, following intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT) in neonatal rats. TPH activity in central serotonergic cell bodies and terminals was reduced to 20-30% of control levels at 1-8 weeks after neonatal, low-dose 5,7-DHT injection (24 micrograms free base). In contrast, AADC activity was either not changed or decreased to 40% of control levels, depending on the region. In situ hybridization and immunocytochemical staining indicated that 5,7-DHT caused a marked reduction in TPH and AADC message levels as well as the number of 5-HT and AADC-immunoreactive cells within the DRN as early as 1 week after 5,7-DHT. Even 15 weeks after drug administration recovery did not occur. This apparent neuronal loss was region-specific suggesting that some serotonergic neurons are more resistant to neonatal 5,7-DHT treatment than others. Taken together, these studies indicate that neonatal treatment with 5,7-DHT produces a marked and permanent (up to 15 weeks) reduction in the number of central serotonergic neurons.

5,7-Dihydroxytryptamine↗
PubMed1993

Bromodeoxyuridine induces chromosomal fragile sites in the canine genome.

Peripheral blood lymphocytes from 3 clinically normal domestic dogs were cultured for bromodeoxyuridine (BrdU) induction of fragile site expression. BrdU induced fragile site expression in cells from all 3 dogs. The mean percent of cells with fragile sites and the mean number of fragile sites per cell were significantly increased in all BrdU incubated cultures compared to control cultures. The frequency of BrdU fragile site expression did not vary significantly among the dogs. Lymphocytes from all 3 dogs expressed BrdU induced autosomal fragile sites. Two BrdU induced fragile sites were identified on the long arm of chromosome 1, one of which was close to or coincident with a previously identified folate sensitive fragile site on this canine chromosome. Lymphocytes from the 2 female dogs also expressed BrdU induced fragile sites on the X chromosome, but BrdU failed to induce fragile sites on the X chromosome from the one male dog in the study. The 2 BrdU-induced fragile sites identified on the long arm of the X chromosome were close to, or coincident with 2 previously described folate-sensitive common fragile sites on the canine X chromosome. This is the first report of induction of BrdU-inducible fragile sites in the genome of the domestic dog.

Animals↗