Biomedical subjects

A C Webb

Publications and source records attributed to A C Webb.

At least 19 recordsLinked to original sources

PubMed1992

Morphological correlates of protein kinase C induced potentiation in the chick brain slice.

Continuous perfusion of a chick brain slice with 10 microM 4 beta phorbol 12,13 diacetate (PDAc) produces a significant increase in the amplitude of the response to electrical stimulation at 0.1 Hz recorded within the intermediate and medial part of the hyperstriatum ventrale (IMHV). This PDAc-induced potentiation and that induced by tetanising stimulation appear to share similar mechanisms. Quantitative electron microscopy of synapses within the IMHV from slices in which a PDAc induced potentiation had been produced 30 minutes earlier showed that compared with control slices there was a significant increase in the size of the postsynaptic density of spine synapses. The change was greater in magnitude than that observed in spine synapses following tetanically induced potentiation. No other synaptic parameters were affected.

Animals↗

PubMed1992

Protein kinase activity and synaptic plasticity in a chick brain slice.

In an in vitro slice preparation of the chick brain it is possible to record responses to single electrical stimuli from within the intermediate and medial part of the hyperstriatum ventrale (IMHV), a region known to be involved in learning. The amplitude of such responses is significantly increased by superfusion of the slice with 10 microM 4 beta-phorbol 12,13 diacetate (PDAc), a phorbol ester which stimulates protein kinase activity. The ability of PDAc to induce potentiation is greatest in chicks less than 6 days old. Administration of the kinase antagonist H7 prevents the induction of persistent potentiation and in fact produces a long lasting depression of response amplitude. H7 also produces a short term increase in excitability within the IMHV and results in increased expression of N-methyl-D-aspartate receptor activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation.

Animals↗

PubMed1992

The immediate early genes of human cytomegalovirus require only proximal promoter elements to upregulate expression of interleukin-1 beta.

Human cytomegalovirus (HCMV) can infect monocytes and macrophages. The immediate early one (IE1) gene product of HCMV positively regulates its own expression, as well as the expression of the interleukin-1 beta (IL-1) gene. This study describes the IL-1 promoter proximal region required for upregulation of IL-1 gene expression by the HCMV IE1 or IE1 plus IE2 gene products. An IL-1 chloramphenicol acetyltransferase (CAT) construct containing the IL-1 genomic upstream sequence from position -1097 to +14 and four additional IL-1CAT plasmids containing progressive deletions of the -1097 to -131 sequence were used to evaluate the effect of the HCMV IE gene products on IL-1 gene expression. IL-1CAT plasmids were transfected into a monocytic cell line, THP-1, with plasmids containing either the IE promoter-regulatory region upstream of the bona fide IE1 (pIE1), IE2 (pIE2), or IE1+2 genes (pIE1+2) or a control plasmid containing the IE promoter-regulatory region alone (pLink760). In the presence of pIE1+2, there was an approximate 15-fold increase in CAT activity compared with the control, pLink760, in cells with CAT plasmids containing the -1097 to +14 IL-1 sequence. Plasmids with progressive deletions of this sequence, including the plasmid containing the shortest upstream segment (-131 to +14) also had an approximate 15-fold increase in CAT activity. The upregulation of IL-1 expression was mediated, primarily, by IE1 and not by IE2. This effect was promoter specific because an IL-1CAT plasmid with a complete deletion of the proximal promoter elements (-234 to +146) did not respond to the HCMV IE gene products.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloramphenicol O-Acetyltransferase↗

PubMed1991

Potentiation of synaptic responses in slices from the chick forebrain.

Coronal slices, containing part of the medial hyperstriatum ventrale (MHV), were cut from the left forebrains of domestic chicks and maintained in vitro. Records were made of the field responses evoked in the MHV by local electrical stimuli provided at 0.1 Hz. Two 1 min periods of stimulation at 5 Hz, separated by 10 min, were used in attempts to induce a persistent increase in the size of the postsynaptic response to test stimulation at 0.1 Hz. This procedure produced a potentiation which usually lasted longer than 2 h. The probability of inducing this persistent potentiation of the response (PPR) is not distributed evenly over the whole anteroposterior length of the MHV but is higher in slices that also contain the septo-mesencephalic tract ventrally. These are the slices that contain the intermediate part of the medial hyperstriatum ventrale (IMHV); an area that is essential for early behavioural learning. At this level PPR is not confined to the IMHV. It can also be produced in the lateral neostriatum in response to similar local stimulation at 5 Hz. No PPR was observed in either the caudal ectostriatum, or the paleostriatum.

Animals↗

PubMed1991

The effects of age and visual experience on potentiation of responses in slices from the chick forebrain.

A single coronal slice, containing the intermediate part of the medial hyperstriatum ventrale (IMHV) was cut from the left forebrain of a series of domestic chicks and maintained in vitro. Records were made of field responses evoked in the IMHV by local electrical stimuli. Two 1-min periods of 5 Hz stimulation, separated by 10 min, were used in attempts to induce persistent potentiation of the responses (PPR) to test stimulation at 0.1 Hz. In dark-hatched chicks the probability of producing PPR is much higher in slices from chicks aged 2-5 days post-hatch than in those from either younger or older birds. As an independent measure of plasticity in dark-hatched chicks, the probability of eliciting unit responses to repeated stimulation of remote sites in the slice at 3.3 Hz was analysed. This probability was greater in slices from chicks aged 2-5 days than in those from either younger or older birds. In light-hatched chicks the probability of inducing PPR is significantly higher during the first day post-hatch, than in dark-hatched chicks of this age. The probability of producing PPR in slices from light-hatched chicks aged 2-3 days is less than that in slices from either younger or older birds. It is clear that both the age and past experience of the domestic chick affect the neurophysiological properties of slices of brain, tested in vitro.

Age Factors↗

PubMed1991

Morphological correlates of persistent potentiation in the chick brain slice.

In an in-vitro slice preparation of the chick brain it is possible to induce persistent potentiation of responses to single electrical stimuli by giving two bursts of 300 stimuli at 5 Hz separated by ten minutes of control stimulation at 0.1 Hz. We investigated the morphological correlates of this potentiation in a group of 2 day old chicks using quantitative electron microscopical techniques. It was found that in slices which showed a clearly potentiated response there was a significant increase in the size of the postsynaptic densities of synapses on spines in the left hyperstriatum ventrale (IMHV). No such increases were seen in a control group nor in slices which failed to potentiate. These results provide further evidence for the lability of synapses in the IMHV.

Animals↗

PubMed1991

Use of the polymerase chain reaction for screening and evaluation of recombinant baculovirus clones.

We report the application of the PCR for screening and high-resolution characterization of recombinant baculovirus clones. Starting with less than 10 nanograms of viral DNA, it is possible to 1) demonstrate that the DNA sequence to be expressed has not been deleted or rearranged during the co-transfection or homologous recombination events, 2) test for the presence of wild-type virus in the isolate and 3) generate amplified DNA that can be used for nucleotide sequence analysis or high-resolution restriction analysis. The method is based upon PCR of genomic viral DNA prepared from primary amplified stocks of extracellular virus using a small-scale procedure. The approach has special relevance for definitive characterization of recombinant virus used to express point mutant proteins and for characterization of recombinant virus generated through use of mixed oligonucleotides or random mutagenesis.

Animals↗

PubMed1990

Local circuitry in the IMHV of the domestic chick (Gallus domesticus).

The responses to local stimulation have been recorded from neurons in the intermediate part of the medial hyperstriatum ventrale (IMHV) of the domestic chick, by using an in vitro slice preparation. When the slice is bathed in gassed Krebs' solution, a single stimulus evokes a short-lasting diphasic response. The first phase is negative and lasts some 3 ms, whereas the second, positive phase is often of lower amplitude and usually persists for about 15 ms. The first phase is little altered by perfusion with either Ca2(+)-free Krebs' solution or Krebs' solution containing a high concentration of Mg2+. In contrast, the second phase is abolished by these procedures. The post-synaptic phase is positive when it is recorded anywhere between 0.1-1.25 mm from the stimulated point; however, in the immediate vicinity (0.0-0.1 mm) of the stimulating electrodes, the post-synaptic response is strongly negative. A pair of stimuli has to be separated by at least 10 s to guarantee complete recovery of excitability of the post-synaptic response. The recovery curve for this response shows a refractory period of some 5 ms, a peak of excitability at an interval of about 20 ms, and then a sharp trough of relative inexcitability at about 200 ms. The post-synaptic response is considerably reduced in magnitude and duration by the addition of AP-5 to the perfusion fluid; the remaining post-synaptic response is completely abolished by kynurenic acid. The addition of bicuculline methiodide in concentrations of at least 1 x 10(-6) M increases both the magnitude and duration of the second, positive phase of the response to single stimuli. This extended positive response (which may last from 500-800 ms) is abolished by perfusion with bicuculline dissolved in Ca2(+)-free Krebs' solution. For the entire duration of the extended post-synaptic positive response produced by bicuculline, the irregular discharge of single neurons can be recorded. Like the post-synaptic positive response in Krebs' solution, the much larger response produced by bicuculline shows a very localized negativity beneath the stimulating electrodes and displays an almost identical time-course for the recovery of excitability following a single stimulus. The bicuculline induced positive response is also considerably reduced by the presence of AP-5; the addition of kynurenic acid abolishes the remaining post-synaptic response completely. A post-synaptic response, similar to that produced under bicuculline, can be produced by the addition of a maximally effective dose of d-tubocurarine.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1990

Connections of the IMHV in the domestic chick Gallus domesticus.

The responses to single electrical stimuli have been recorded from neurons in the brains of domestic chicks, by using an in vitro preparation consisting of a coronal slice taken from the forebrain. All slices were cut so that they contained the intermediate part of the medial hyperstriatum ventrale (IMHV). When such a slice is bathed in standard Krebs' solution there is no evidence that the excitation produced by a single stimulus can be transmitted more than 1 mm either towards or away from the IMHV. The addition of bicuculline methiodide (more than 3 x 10(-6) M) to Krebs' solution allows the excitation produced by a single stimulus to spread in all directions throughout the dorsal half of a coronal slice. At points remote (more than 1.5 mm) from the stimulated point, the magnitude of the spreading wave of excitation bears an all-or-nothing relation to the strength of stimulus used to excite it. This wave of excitation spreads from the excited point in all directions without attenuation at 0.9 +/- 0.017 (s.d.) m s-1 and consists of a prolonged burst of activity of the invaded neurons. The properties of coronal slices described above are also true of brain slices cut in a parasagittal plane. The spreading response to a single stimulus given in the presence of bicuculline, can be reduced in magnitude by the addition of AP-5 but it still spreads throughout the dorsal part of the slice at the same velocity. The response can be eliminated by the addition of kynurenic acid. The addition of curare to the bathing medium produces similar responses that spread in a similar fashion to those seen under bicuculline. These results suggest that the dorsal part of the forebrain of the domestic chick (in fact, the part derived from the embryological alar plate) contains a network of reciprocally connected local circuits. Transmission throughout the network is normally prevented by active inhibition.

Animals↗

PubMed1990

Chromosomal organization and localization of the human urokinase inhibitor gene: perfect structural conservation with ovalbumin.

Plasminogen activator inhibitor 2 (PAI-2) plays an essential role in the regulation of localized extracellular proteolysis by its inactivation of urokinase. Using probes derived from a cDNA we isolated from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes, we have mapped, isolated, and determined the molecular organization of the gene for PAI-2 (PLANH2). In situ hybridization of the cDNA to normal metaphase chromosomes has confirmed our prior assignment of the gene for PAI-2 to chromosome 18 and further localized it to the long arm at 18q21.2-18q22. We have isolated nine independent genomic clones, two of which were found to contain the entire PAI-2 transcriptional unit of approximately 16.4 kilobase pairs (kbp). Analysis of the gene organization by restriction enzyme mapping, Southern blotting, and DNA sequencing revealed that the cDNA sequence is divided among eight exons interrupted by seven introns, the junctions of which all conform to the "GT-AG" consensus rule. In common with the arrangement found throughout, the serpin superfamily, of which PAI-2 is a member, the first intron is located just 5' to the initiator methionine residue, and the 3' untranslated region (UTR) is not interrupted by a splice junction. Determination of the transcription initiation site by primer extension analysis of monocytic mRNA indicated that our PAI-2 cDNA was, at most, only three nucleotides short of full length, yielding a primary PAI-2 transcript with a 66-bp first exon. A promoter "TATAAAbox" is located 30 bp upstream of the "cap" site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Identification of interleukin-1 in equine osteoarthritic joint effusions.

Interleukin-1 (IL-1) is a protein secreted by stimulated cells of the monocyte-macrophage line, which has a number of important biologic activities. Interleukin-1 has been implicated in the induction and augmentation of the pathologic processes involved in arthritis and articular cartilage destruction. Horses develop osteoarthritis with a frequency and degree of severity similar to human beings. To further document the similarity of the osteoarthritic process in people and horses, the synovial fluid from 5 horses with clinical osteoarthritis was tested for IL-1 bioactivity. Interleukin-1 activity was found in all tested synovial fluids. Upon column chromatography, the synovial fluid-derived factor had a molecular weight consistent with that of IL-1 in other mammalian species. Ion exchange chromatography of osteoarthritic synovial fluid revealed the principal peaks of bioactivity to be in the fractions with isoelectric points of 7.2, 5.4, and 4.7, which are characteristic of IL-1. A considerable degree of homology between human and equine IL-1 was demonstrated by the cross hybridization of human IL-1 beta cDNA probe with RNA derived from IL-1-producing equine adherent monocytes. These results indicate that equine IL-1 is in all of the osteoarthritic equine joints tested and that equine IL-1 has many of the characteristics of IL-1 isolated from other species.

Animals↗

PubMed1989

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone.

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.

Animals↗

PubMed1988

Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides.

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.

Chromosome Deletion↗

PubMed1988

Modulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-2 expression by U-937 mononuclear phagocytes. Effects of 1 alpha, 25-dihydroxyvitamin D3 and phorbol ester.

Mononuclear phagocytes are known to produce both urokinase-type plasminogen activator (u-PA) and a specific PA inhibitor, PAI-2. In this study we have investigated the effects of calcitriol and PMA-induced differentiation on the comparative expression of PA and PAI in monoblast-like U937 cells. Cells were incubated for 96 h in the presence or absence of 50 nM calcitriol. After transfer into serum-free medium, the cells were cultured for 48 h with PMA (0 to 50 ng/ml). PA and PAI activities of conditioned media and cell lysates were measured with a plasminogen dependent colorimetric assay. Control cells uniformly secreted PAI activity (70.3 +/- 24.9 PAI U/ml), while calcitriol pretreatment induced the cells to secrete PA activity (52.6 +/- 47.2 milli-Ploug unit/ml). PMA induced secretion of PAI activity in calcitriol-pretreated cells to levels 4.6- to 8.3-fold greater than controls (p less than 0.05). Parallel effects on PA and PAI activities were seen in cell lysates. To determine how changes in the expression of u-PA and PAI-2 might account for these effects, mRNA for u-PA and PAI-2 were assessed by Northern blot analysis. Calcitriol induced an increase in u-PA mRNA with a marked reduction in PAI-2 mRNA. PMA alone induced modest increases in both mRNA species. In calcitriol pre-treated cells, PMA induced a moderate increase in u-PA mRNA and a marked increase in PAI-2 mRNA. We conclude that agonist-specific differentiation of U937 cells modulates the expression of PA and PAI activities by altering the proportionate biosynthesis of u-PA and PAI-2 proteins. The ability of mononuclear phagocytes to control plasminogen activation at inflammatory foci may therefore be contingent on the independent regulation of u-PA and PAI-2 gene expression.

Blotting, Northern↗

PubMed1988

Response characteristics of neurons in chick forebrain slices.

Coronal sections were taken from the forebrains of domestic chicks, aged 1-20 days, and maintained in vitro. Extracellular recordings of neural activity were made from the intermediate part of the medial hyperstriatum ventrale (IMHV). Spontaneous activity was rarely recorded, but neuronal responses could be evoked by stimulation of various sites. Each recording point was surrounded by an arc of sites which, when stimulated, typically elicited a short-latency field potential. These 'local responses' could be recorded in slices from chicks of any age. Stimulation of more distant sites failed to evoke field potentials from the IMHV. Instead, trains of large, unit action potentials appeared on an undisturbed baseline. Such 'unit' responses could only be evoked by stimuli delivered at specific frequencies. They required facilitation, were of variable latency, and often finally decayed. The number of sites capable of evoking a 'unit' response from the IMHV fell dramatically in slices taken from chicks older than 4 days.

Aging↗

PubMed1988

Human pro-IL-1 beta gene expression in monocytic cells is regulated by two distinct pathways.

We have previously reported that the pro-IL-1 beta gene is transiently expressed in THP-1 human monocytic leukemia cells after stimulation with bacterial LPS. Herein we show differential pro-IL-1 beta gene transcription in the same cell type using two distinct stimuli. This transcriptional difference is also reflected at the level of intracellular IL-1 beta protein production. In contrast to LPS, a phorbol ester (PMA) induces a stable, non-transient population of mRNA. Furthermore, each induction pathway is operational under conditions where the other is inhibited, suggesting functional independence. Evidence is presented for post-transcriptional regulation of the two responses in THP-1 cells at the level of mRNA stability. We also demonstrate a similar dualistic response in primary monocyte-derived human macrophages as well as the human myelocytic cell line HL-60.

Cell Line↗

PubMed1988

Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)+ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambda gt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0-kilobase cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humoral syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.

Amino Acid Sequence↗