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Biomedical subjects

A C Webb

Publications and source records attributed to A C Webb.

At least 55 records · Page 3Linked to original sources

Transmission of burst responses through slices of rat cerebral cortex.

1. Slices of rat's forebrain, 400 micron thick, have been cut and maintained in a bath perfused with warm oxygenated Krebs solution. Records were made with extracellular micropipettes of the neural responses to local stimulation of the cortex itself or the underlying white matter. 2. Single stimuli at either of these sites could produce an all-or-nothing burst response among nearby neurones. This response usually lasted 0.2-0.5 s during which repetitively discharging cortical units could be recorded at all cortical depths. 3. This burst response was transmitted from the stimulated point across the cortex in all directions with a velocity of roughly 0.1 m s-1. 4. Complete recovery of excitability among neurones generating the burst response took about 10 s. 5. Removal of Ca2+ from the perfusate prevented transmission of this response, as did a high concentration of Mg2+ or 160 mg/100 ml of ethanol. 6. Propagation of the burst response was not dependent upon the integrity of the underlying white matter; it required only that any 20% of the cortical thickness was intact and undamaged. 7. In coronal sections of brain the response could be transmitted from one hemisphere to the other provided that the corpus callosum was intact.

Action Potentials↗

Identification of transcripts encoding a parathyroid hormone-like peptide in messenger RNAs from a variety of human and animal tumors associated with humoral hypercalcemia of malignancy.

The syndrome of humoral hypercalcemia of malignancy (HHM) appears to be mediated in many instances by a parathyroid hormone-like peptide, which has recently been purified, sequenced, and cloned. Using a probe representing the coding region of the human PTH-like peptide, we examined by Northern analysis poly (A)+ RNA from a variety of human and animal tumors associated with HHM. Hybridizing transcripts were identified in mRNA from each of 12 human and each of four animal HHM-associated tumors, with a complex hybridization pattern observed in the human mRNAs and a relatively simple pattern observed in the animal mRNAs. Poly (A)+ RNA prepared from tumors of similar histological types unassociated with HHM failed to hybridize with the probe. Messenger RNA-dependent biological activity from the animal tumors was entirely eliminated in a hybridization-arrest experiment using a complementary oligonucleotide spanning the region of homology between human PTH and the PTH-like peptide. These findings indicate that the PTH-like peptide is associated with the syndrome of HHM in a wide spectrum of tumor types from a variety of mammalian species and that the PTH-like sequence in the proximal amino terminus of the peptide is highly conserved.

Amino Acid Sequence↗

Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency.

The chain-breaking (peroxyl radical-trapping) antioxidant activity of plasma obtained from several patients with a very severe vitamin E deficiency has been measured. The total chain-breaking antioxidant activity in lipid extracts has been shown to be approximately equal to the concentration of vitamin E. For whole plasma there is no significant difference in the concentrations of water-soluble, chain-breaking antioxidants between the E-deficient patients and healthy adults. It is concluded that even in cases of very severe vitamin E deficiency the requirement for this vitamin is not met by some other exogenous or endogenous antioxidant.

Antioxidants↗

Human monocyte Arg-Serpin cDNA. Sequence, chromosomal assignment, and homology to plasminogen activator-inhibitor.

An LPS-stimulated, human monocyte cDNA library was screened for stimulation-specific clones. One clone (pcD-1214) contained a 1.9-kb pair insert that hybridized to a 2,000-nucleotide mRNA expressed by peripheral blood monocytes, the histiocytic lymphoma cell line U937, and umbilical cord endothelial cells. The 415-amino-acid precursor polypeptide predicted from the cDNA (46,596 molecular weight) has a putative 22-residue signal peptide and approximately 35% homology with members of the serine protease inhibitor (Serpin) superfamily. On the basis of amino acid homology and alignment of COOH-terminal residues within the Serpin-reactive center, the clone pcD-1214 was identified as coding for an Arg-Serpin. Southern blot analysis of human-mouse somatic cell hybrid DNA locates the Arg-Serpin gene on human chromosome 18. A perfect match between amino acid residues 347-376 in this Arg-Serpin and the published sequence of a 30-residue, tryptic peptide from the COOH-terminus of a monocyte plasminogen activator-inhibitor (PAI-2), strongly suggests that the Arg-Serpin encoded by pcD-1214 is PAI-2.

Amino Acid Sequence↗

Transcriptional regulation of the human prointerleukin 1 beta gene.

Interleukin 1 (IL 1) is a protein produced by monocytes in response to certain antigens which produces a wide variety of cellular responses in various tissues. We have studied the regulation of the human proIL-1 beta gene in THP-1 human monocytic leukemia cells. Lipopolysaccharide (LPS) induction of this gene results in an immediate and transient increase of message that rapidly falls to a low, but constant, level within 6 hr. This decrease results from a specific repression of transcription by 2 hr after stimulation. Cycloheximide inhibition of new protein synthesis causes a superinduction of IL 1 message, but does not alter the initial kinetics of message production. This presumably delays the synthesis of a labile transcriptional repressor protein and implies that the proIL-1 beta gene is under the control of both a transcriptional activator and a newly synthesized transcriptional repressor. The transient increase in mRNA production and the sustained low-level synthesis beyond the initial transient response suggest that the IL 1 protein itself may act intracellularly in a manner analogous to that described for several proto-oncogenes and cellular competence factors.

Cell Line↗

Studies on the molecular nature of human interleukin 1.

Adherent human blood monocytes were stimulated with heat-killed Staphylococcus albus or Escherichia coli lipopolysaccharide in the presence of 35S-methionine-, [3H]leucine-, or 14C-labeled amino acids. After incubation, interleukin 1 (IL 1) activity in the supernatant medium was purified over an anti-human IL 1 immunoadsorbent followed by gel filtration and chromatofocusing. The purity of the IL 1 was assessed by fluorography of one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Isoelectric and chromatofocusing of low m.w. proteins (less than 20,000 m.w.) revealed three charged 18,000 m.w. species of IL 1 with approximate pI's of 7, 6, and 5, with the most abundant form at pI 7. During the purification procedures, lymphocyte co-mitogenic activity, fever in rabbits, and prostaglandin E2 release from dermal fibroblasts co-eluted in the same fractions. In addition, these fractions were active when injected into endotoxin-resistant C3H/HeJ mice for the production of fever, the induction of serum amyloid A protein, a decrease in serum iron concentration, and an increase in the number of circulating neutrophils. Fluorography revealed homogeneous bands with an m.w. of about 18,000 which correlated with these biological activities. The specific activity of the pI 6 or 5 IL 1, as judged by the ratio of T cell co-mitogenic activity to incorporated radiolabeled amino acid, was at least 10-fold greater than that observed for the pI 7 form. This result suggests that the amino acid compositions of the two 18,000 m.w. acidic forms are unrelated to the pI 7 species. These results also demonstrate that the pI 7 human monocyte IL 1 is the predominant 18,000 m.w. form synthesized and, furthermore, that homogeneous pI 7 IL 1 exhibits multiple biological properties on various tissues by modulating immunologic, inflammatory, metabolic, and neurologic functions. Data are also presented for the existence of a high m.w. (32,000) human pro-IL 1 molecule as the predominant monocytic intracellular form. This pro-IL 1 is degraded artifactually during isolation to lower m.w. forms in the presence of an extracellular serine protease activity. These data are consistent with a model for IL 1 secretion in which pro-IL 1 is first synthesized within the cell and is processed during or after extracellular transport.

Biological Assay↗

The effects of arginine-8 vasopressin on blood vessels supplying the cerebral cortex.

A videocamera and a dissecting microscope have been used to record the effects of arginine-8 vasopressin (AVP) upon pial blood vessels in anaesthetised rats. Topical application of AVP caused a contraction of pial arteries, but had no measureable effect upon the diameter of veins. The smallest concentration of AVP that was effective in contracting arteries was 10(-7) mU/microliter. Stronger solutions (10(-5) to 2.0 mU/microliter) produced approximately the same (45%) reduction of external diameter. Contraction was maximal 0.25-1.0 min after application of the hormone, had almost recovered (10% contraction) after 10 min, and showed complete recovery by 30 min. Concentrations of AVP that were greater than 10(-3) mU/microliter produced tachyphylaxis, so that a second application of AVP 30 min later had considerably less effect. Concentrations less than 10(-3) mU/microliter produced no detectable tachyphylaxis. These results suggest that blood flow to the normal cerebral cortex may be partly under tonic control by the local concentration of AVP.

Animals↗

Genomic sequence for human prointerleukin 1 beta: possible evolution from a reverse transcribed prointerleukin 1 alpha gene.

We have isolated the human prointerleukin 1 (proIL-1) beta gene from leukocyte and fetal liver libraries. The nucleotide sequence and its gene organization reveals that the proIL-1 beta gene is composed of seven exons with a primary transcription product length of 7,008 nucleotides. The exon sequence agrees well with that of the human proIL-1 beta cDNA. Features of interest within the transcriptional unit include positioned TATA, CAT, and poly-adenylation signals for gene regulation, as well as the signatures of gene duplication via retrotransposition in the form of flanking direct repeats and a genomic poly A tail. The genomic organization of the proIL-1 beta gene with respect to the number and position of exon boundaries is strikingly similar to that of the recently reported human proIL-1 alpha gene. Therefore, we hypothesize that the proIL-1 beta may have arisen by a reverse transcriptase mediated duplication of the related alpha gene.

Base Sequence↗

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.

Animals↗

The effects of arginine-8 vasopressin on the cerebral cortex of rats anaesthetised with urethane.

The effects of arginine-8 vasopressin (AVP) on the spontaneous electrical activity of the cerebral cortex were investigated in rats anaesthetised with urethane. Direct application of a relatively large concentration of AVP (0.5-2.0 mU AVP/microliter; 1.25-5.00 ng AVP/microliter) to the pial surface of a small area of the parietal cortex produced a complete cessation of spontaneous neural activity, which was often preceded by a short-lasting increase in frequency of discharge. There was also a decline in the number of large waves in the local electrocorticogram. These changes occurred after 1-4 min. A slow recovery began 10-30 min later, but was sometimes still incomplete after more than an hour. Only the first application of AVP produced the local effects described above. A second application, 2 hr later, was without effect. Direct application to a small area of one hemisphere did not alter the electrical activity of the contralateral cortex. Smaller concentrations of AVP (0.005-0.200 mU AVP/microliter; 12.5 pg-0.5 ng AVP/microliter) were variable in their actions. Occasionally, no effect at all was seen, while sometimes the cells stopped firing completely. However, the most usual consequence was a reduction in discharge frequency, the magnitude of which appeared to be related to the concentration of AVP applied. A second application of a small concentration of AVP usually failed to reduce the rate of spontaneous discharge.

Animals↗

Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.

"Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor.

Animals↗

Multiple biological activities of human recombinant interleukin 1.

Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever.

Amino Acids↗

Interleukin-1 gene (IL1) assigned to long arm of human chromosome 2.

A complementary DNA (cDNA) probe for the predominant (pI 7) form of human monocyte-derived interleukin-1 (IL1) and a collection of 30 human-mouse somatic cell hybrids were used to assign the IL1 gene to human chromosome 2 by Southern blot analysis of hybrid cell DNA digested with the restriction endonuclease BglII. In situ hybridization to human metaphase chromosomes localized the IL1 gene to the long arm of chromosome 2 at position 2q13-2q21 between two fragile sites.

Animals↗

Messenger ribonucleic acid from tumors associated with humoral hypercalcemia of malignancy directs the synthesis of a secretory parathyroid hormone-like peptide.

Previous studies have provided evidence that tumors associated with humoral hypercalcemia of malignancy produce a factor that shares certain biological properties with, but is not, native PTH. In the present study, media from Xenopus oocytes microinjected with polyadenylated RNA prepared from three human or animal tumors associated with humoral hypercalcemia of malignancy are shown to have activity in the cytochemical bioassay for PTH. This activity parallels that of the PTH standard in the assay and is completely inhibited by the PTH analog [Nle8,Nle18,Tyr34]bovine PTH-(3-34)NH2. Media from oocytes injected with mRNA from control tumors contain no such activity. The mRNA encoding this activity in one of the animal tumors has been enriched approximately 10-fold by preparative polyacrylamide gel electrophoresis. These results demonstrate that tumors associated with humoral hypercalcemia of malignancy have the capacity for directing the synthesis of a secretory protein that fulfills certain criteria for being the humoral mediator in question. These techniques may provide the basis for molecular cloning of this factor.

Animals↗

Human and murine interleukin 1 possess sequence and structural similarities.

The molecular cloning and sequence analysis for human and murine interleukin 1 precursor have recently been described. Comparison of the amino acid sequences resulting from these data can be used to aid in the identification of conserved regions essential to biological activity. We report results which confirm the relationship between these two molecules and suggest that specific regions may be essential for activity. Amino terminal sequence analysis of a 19,000 Mr biologically active IL-1 isolated from stimulated human monocytes reveals a sequence which is in good agreement with that inferred from the human cDNA and, furthermore, locates the processed amino terminus at a site similar to that described for the murine sequence.

Amino Acid Sequence↗

Nucleotide sequence of human monocyte interleukin 1 precursor cDNA.

Interleukin 1 (IL-1) is a protein with several biological activities regulating host defense and immune responses. We report here the isolation of human IL-1 cDNA. It encodes a precursor polypeptide of 269 amino acids (30,747 Mr). mRNA isolated by hybridization to this cDNA was translated in a reticulocyte cell-free system, yielding immunoprecipitable IL-1. Furthermore, this hybrid-selected mRNA was injected into Xenopus laevis oocytes, which subsequently secreted biologically active IL-1. The cDNA nucleotide sequence suggests that IL-1 is initially translated as a precursor molecule that is subsequently processed into the 15,000-20,000 Mr protein usually associated with IL-1 activity.

Amino Acid Sequence↗