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Biomedical subjects

D J Price

Publications and source records attributed to D J Price.

At least 19 recordsLinked to original sources
PubMed1992

Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase.

The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.

Animals↗
PubMed1992

Evidence that the earliest generated cells of the murine cerebral cortex form a transient population in the subplate and marginal zone.

We used bromodeoxyuridine to label the earliest generated cells of the murine cerebral cortex while they were dividing, and then observed their distributions at several instances later in development. Shortly before birth, many of the labelled cells were either above the cortical plate, in the marginal zone, or below it, in the region known as the subplate in other species. These cells had disappeared by postnatal day 21.

Animals↗
PubMed1992

Organization of association projections from area 17 to areas 18 and 19 and to suprasylvian areas in the cat's visual cortex.

Cells in area 17 that are labelled by single, discrete injections of retrogradely transported tracers into extrastriate visual areas are discontinuously distributed in dense patches. In this study we made multiple, closely spaced injections of fluorescent dyes into extrastriate areas, to generate large deposits that would reveal whether the distributions of corticocortical cell bodies in area 17 are truly patchy or appear clustered only after small injections. By injecting a different tracer into each extrastriate area, or group of areas, we examined the spatial relationships between the populations of association cells. All deposits of tracers in areas 18, 19, or suprasylvian cortex, irrespective of size, label cells in a series of clusters in topographically related parts of area 17. We conclude that the complete populations of cells in area 17 that project to areas 18, 19, and the lateral suprasylvian cortex are all genuinely distributed in a patchy fashion. There appears to be a complex relationship between the sets of association cells projecting to different extrastriate regions: they do not completely overlap, only partially, and share some cortical zones but not others. In these experiments, only tiny percentages (2-5%) of labelled cells in the overlapping regions were filled with both tracers, suggesting that very few association cells in area 17 project to more than one of the extrastriate areas we studied. By comparing the dimensions of each injection site and of the labelled region in area 17, we estimated the extent of the convergence from area 17 to areas 18, 19, and posteromedial suprasylvian areas in retinotopic terms. The functional convergence was very similar in these pathways.

Amidines↗
PubMed1992

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.

Amino Acid Sequence↗
PubMed1991

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase.

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.

Amino Acid Sequence↗
PubMed1991

Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II.

The insulin-stimulated protein kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase II. The substrate specificities of ISPK and S6 kinase II were also very similar and like S6 kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of a leech homolog to the Drosophila engrailed gene.

We have cloned and sequenced a homolog (ht-en) to the Drosophila engrailed (en) gene from the glossiphoniid leech, Helobdella triserialis. Amino acid comparisons of the ht-en homeodomain and C-terminal residues with the corresponding residues encoded by en-class genes of other species reveal 75-79% sequence identity. In addition, the ht-en sequence appears to have a serine-rich region 16 residues C-terminal from the homeodomain, which by analogy to Drosophila may be a target site for phosphorylation. The leech gene encodes some amino acid substitutions for residues that are highly conserved in other species. These are found within the second and third of the three putative helices of the homeodomain, and in both of the intervening turn regions.

Amino Acid Sequence↗
PubMed1991

Susceptibility of captive wildfowl to avian tuberculosis: the importance of genetic and environmental factors.

This study reports the findings of an epidemiological survey of death due to avian tuberculosis in the captive collection of wildfowl at The Wildfowl and Wetlands Trust Centre, Slimbridge, Gloucestershire. Both genetic and environmental factors have been shown to affect the incidence of, and the birds' susceptibility to, the disease. Seasonal body condition was related to the occurrence of death due to the disease in both males and females. Birds from either hot or cold climates appeared to have a higher incidence than those from temperate climates. What the birds ate did not affect incidence but the method they used for obtaining their food did. Higher susceptibility was found in those species evolved for marine or arboreal habitats. Anomalies in susceptibility which suggest a higher level of genetic immunity in some groups have also been found. Reasons are put forward to explain these findings.

Animals↗
PubMed1991

Sonoluminescence in water and agar gels during irradiation with 0.75 MHz continuous-wave ultrasound.

Free radicals, detected as light emissions (sonoluminescence), can be produced in both simple aqueous systems and agar gels by irradiation with 0.75 MHz continuous-wave (CW) ultrasound using acoustic pressures as low as 200 KPa. Although the acoustic pressures necessary for free radical formation in tap water gels (200 KPa), are considerably higher than those required for the formation of macroscopic visible bubbles (26 KPa), the sonoluminescence threshold (0.5 W/cm-2 SATA equivalent) falls at the lower end of the intensity range (0.5-3 W/cm-2) commonly used by ultrasonic physiotherapy equipment. In respect of the overall features of cavitation processes, agar gels appear to be suitable simple systems in which to model cavitation in vivo. However, cavitation processes leading to sonoluminescence are themselves complex as illustrated by the findings that small changes in ionic composition or pH have a significant effect on both the acoustic pressure threshold and the extent of sonoluminescence above this threshold.

Agar↗
PubMed1991

Regressive changes among corticocortical neurons projecting from the lateral suprasylvian cortex to area 18 of the kitten's visual cortex.

The postnatal development of corticocortical neurons projecting from the medial bank of the lateral suprasylvian cortex to area 18 of the kitten's visual cortex was examined using retrograde fluorescent tracers. Area 18 was injected in young kittens aged nine days or less and in older kittens aged 30 days or more. Many of the injected kittens were perfused with fixative four to five days later, but some of the youngest were killed after longer survival periods of 35-50 days (long-survival animals). Labelled neurons in the medial bank of the lateral suprasylvian cortex were densely distributed in both superficial layers (II and III) and deep layers (V and VI) in the kittens injected less than nine days postnatal, irrespective of whether survival was short or long, but they were found almost exclusively in layers V and VI in the old, short-survival animals. Only in the group of old kittens did we find a clear topographical arrangement of projections in the rostrocaudal direction and a correlation between the rostrocaudal lengths of the injection sites and labelled areas. In the other two groups, for a similarly sized injection site, the labelled areas were much longer rostrocaudally than in the old, short-survival kittens, and occupied roughly the posterior two-thirds of the medial bank of the lateral suprasylvian cortex, irrespective of the positions of the injections. In the frontal plane, topography was unclear in all groups. These findings demonstrate that there is considerable postnatal refinement of the projection from the medial bank of the lateral suprasylvian cortex to area 18. This involves a loss of connections originating from superficial layers and a decrease of convergence with the appearance of topography. Our results from long-survival kittens suggest that most of the early exuberant population of corticocortical neurons projecting from the medial bank of the lateral suprasylvian cortex to area 18 survive beyond the first postnatal month but undergo axonal elimination during this period.

Aging↗
PubMed1991

Effects of general anesthetics and pressure on mammalian excitatory receptors expressed in Xenopus oocytes.

The effects of general anesthetics and pressure on receptors from the mammalian central nervous system have been investigated using oocyte expression techniques. Poly A+ mRNA extracted from rat whole brain was injected into mature Xenopus oocytes producing depolarizing responses to the fast excitatory neurotransmitters NMDA and kainate and the inhibitory neurotransmitters GABA and glycine. An apparatus was constructed to allow agonist dose-response curves to be determined at high pressures using voltage-clamped oocytes. This was used to investigate the excitatory transmitter kainate. It was found that anesthetics depress the current induced by kainate whereas pressure does not appear to affect the responses associated with this transmitter. Furthermore it was found that pressure does not reverse (or modify in any way) the changes in response brought about by application of anesthetics.

Animals↗
PubMed1991

Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

Amino Acid Sequence↗
PubMed1991

Origin of segmental identity in the development of the leech nervous system.

The leech embryo develops its segmental body plan by means of a stereotyped cell lineage. Each hemilateral segment arises from a small set of embryonic blast cells via a comparable sequence of formative cell divisions, and for the most part, lineally homologous cells manifest similar patterns of differentiation in the various hemisegments. Nonetheless, some identified central neurons undergo segment-specific or laterally asymmetric patterns of neuropeptide expression and/or cell death. Certain aspects of this regional diversification result from competitive cell interactions which occur at the level of the postmitotic neuron. However, the neuron's segmental identity is lineally determined, being inherited from its blast cell progenitor over several intervening rounds of mitosis. To learn more about the molecular basis of this phenomenon, we have isolated and begun to characterize leech homeobox genes which are related to the genes that govern segmental identity in other organisms.

Amino Acid Sequence↗