Biomedical subjects
Publications and source records attributed to K Chapman.
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The rat preprotachykinin A (rPPT-A) gene is expressed in restricted populations of neurons and endocrine cells, including a subset of dorsal root ganglion (DRG) neurons. As part of a study to investigate the DNA sequences responsible for tissue-specific expression of the gene, we have determined the sequence of the 5' flanking DNA to 3350 bp upstream of the transcription start site. The sequenced region encodes a number of putative transcription factor binding sites which may play important roles in the regulation of rPPT-A gene transcription.
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The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.
In this study we evaluated the clinical response of 12 patients with malignant melanoma and renal cell carcinoma (RCC) following administration of recombinant human interleukin-2 (rhIL-2) by continuous infusion. Serum samples taken before, during and following sequential courses of IL-2 were assayed for the presence of tumour necrosis factor alpha (TNF-alpha) IL-1 alpha, IL-6 and interferon gamma (IFN-gamma) and the presence or changes in these cytokines were examined with respect to clinical response data: our results did not show any direct correlation between the parameters measured and clinical outcome. In addition, peripheral blood mononuclear cells (PBMC) derived from 3 RCC patients were cultured in a serum-free environment and the resulting supernatants assayed for the production of these cytokines and compared to the corresponding serum levels. During one or more courses of treatment only 1 patient, who had metastatic bone disease, demonstrated detectable serum TNF-alpha; serum IL-6 levels were elevated in a proportion of all patients studied and a sustained IL-6 response occurred in a patient who had complete disease remission; IL-1 alpha was detected in the serum of 3 RCC patients; IFN-gamma could not be detected in any serum sample tested. Cytokine levels in sera and supernatants derived from 3 RCC patients were compared but no correlation was found: TNF-alpha and IL-6 were shown to be present at much higher concentrations in supernatants when compared to sera whereas the levels of IL-1 alpha were almost undetectable. This lack of correlation is probably due to the presence of "interfering" proteins in sera which either depress or enhance the ability to detect cytokines in sera using enzyme immunoassays.
The effects of ethanol and carbon tetrachloride (CCl4) upon tissue vitamin A, liver lipids, liver cytochrome P450 and hepatic morphology were investigated. It was anticipated that CCl4 treatment would have more severe effects upon vitamin A status because CCl4 provides greater hepatic injury than does ethanol. After a 2-week standardization feeding period, young male rats were divided into four groups. For 5 weeks one group of rats (n = 17) received ethanol in liquid diets (30% of calories) while another (n = 8) was exposed to CCl4 inhalation twice a week along with phenobarbital in the diet. All groups received the National Regulatory Commission recommended level for vitamin A. Comparison of ethanol and its pair-fed control group (n = 17) revealed: decreased hepatic vitamin A, no change in serum vitamin A, increased percentage of liver lipid, and cytochrome P450 with moderate fat accumulation in hepatocytes. Comparison of the CCl4-phenobarbital group with pair-fed controls (n = 8) showed: increased serum vitamin A, decreased hepatic vitamin A, increased cytochrome P450, marked hepatic fat accumulation, hepatic cell necrosis, and early cirrhosis. Thus, CCl4 (with phenobarbitol), which is a more potent hepatotoxin as evidenced by a more elevated cytochrome P450 and distorted liver morphology, not only reduced liver vitamin A, but also increased serum vitamin A. The extent of substrate and/or organ specificity remains to be elucidated.
Recombinant human interleukin-6 (IL-6) was assessed for its ability to stimulate bone resorption in prelabeled mouse calvariae in vitro. IL-6 had no effect on bone resorption at concentrations ranging from 300 to 10,000 U/ml (3-1000 pg/ml). Neither the presence of indomethacin nor prolonged incubation periods (96 h) affected this result. IL-6 did not affect resorption stimulated by human recombinant IL-1 alpha (rIL-1 alpha) but inhibited resorption stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. rIL-1 alpha, PTH, and 1,25-(OH)2D3 induced IL-6 release by calvariae. We conclude from these studies that IL-6 does not stimulate bone resorption in neonatal mouse calvariae. However, it may act as a locally produced inhibitor and therefore a paracrine regulator of bone resorption induced by osteotropic hormones. IL-6 could also function as a long-range stimulator of systemic reactions and acute-phase responses to local inflammatory and neoplastic lesions in bone.
The immunological and haematological effects of continuous infusion of recombinant human interleukin-2 (rhIL-2) in 6 patients with metastatic melanoma and 6 with disseminated renal cell carcinoma are reported. In patients with malignant melanoma dacarbazine was given before IL-2; in renal cell carcinoma IL-2 alone was given. In malignant melanoma, 1 complete (CR) and 1 partial response (PR) were seen; 2 patients had stable disease (SD) and 2 progressive disease (PD). In renal cell carcinoma 4 patients had SD and 2 PD. Toxicity of IL-2 therapy was minimal. All patients showed increased cytotoxicity, that was not major histocompatibility complex restricted, towards target cells sensitive and insensitive to natural killer cells. These activities varied between individual patients and were less marked in cases of renal cell carcinoma. Cellular proliferative responses increased in all patients, being consistently higher following the first course of therapy, as did HLA-DR, CD16 and CD25 activation marker expression. Hypersegmentation of neutrophils and eosinophilia were commonly observed, and in renal cell carcinoma these changes were accompanied by abnormal lymphocyte morphology.
The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.
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Interleukin-6 was found to be secreted from seven out of ten human pituitary tumours cultured in vitro. These tumours included prolactin-secreting, growth hormone-secreting and clinically non-functioning adenomas. The amount of Interleukin-6 secreted was variable from 58 to 10,000 units per ml. in medium removed after a four-day incubation under basal conditions. Four tumours secreted IL-6 in excess of 500U/ml, three of which were clinically non-functioning, but each secreted follicle stimulating hormone in vitro. This is the first report of a cytokine growth factor being released basally by human pituitary tumours.
The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures.
The neuropeptides substance P and neurokinin A are synthesised from a family of precursor polypeptides encoded by the preprotachykinin A (PPT) gene. In addition to a mRNA (beta-PPT) containing all 7 exons of the gene, alternatively spliced mRNAs lacking either exon 4 (gamma-PPT) or exon 6 (alpha-PPT) have been identified. We have determined the sequences of cDNA clones encoding four variants of PPT mRNA from rat dorsal root ganglion (DRG), including a novel mRNA species (delta-PPT) in which both exons 4 and 6 are absent. The sequence of delta-PPT predicts the existence of a novel tachykinin precursor polypeptide.
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Recombinant human interleukin-4 (rhIL-4) and transforming growth factor-beta 1 (TGF-beta 1) suppressed the induction of lymphokine-activated killer (LAK) activity induced by recombinant human interleukin-2 (rhIL-2) in peripheral blood lymphocytes. DNA synthesis and the expression of the p55 alpha chain of the IL-2 receptor (Tac antigen) were also inhibited. The inhibitory effect was greatest when these factors were added during the first 48 h of a 4-day culture, with reduced cytolytic activity against both natural killer (NK) resistant and NK-sensitive tumour cell line targets. The suppressive action of both cytokines was accompanied by a reduction in tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in lymphocyte culture supernatants. Recombinant human IFN-gamma (rhIFN-gamma), but not recombinant human TNF-alpha (rhTNF-alpha) was able to overcome the inhibitory effect of recombinant human interleukin-4 (rhIL-4) on LAK induction and DNA synthesis but not Tac antigen expression. However, cytotoxicity induced by rhIFN-gamma alone was also suppressed by rhIL-4 and TGF-beta 1, inferring that rhIFN-gamma-mediated abrogation of rhIL4 suppression was not simply a direct IL-2-independent effect on cytotoxicity. In addition, rhIL-4 did not increase TGF-beta production from rhIL-2-activated peripheral blood mononuclear cells, suggesting that rhIL-4 did not mediate reduction of rhIL-2 responses through the induction of TGF-beta release.
We have identified the chicken equivalent of growth-associated protein GAP-43 in a detergent-resistant membrane skeleton from cultures of chick neurones and embryonic chick brain. Antisera to the membrane skeleton protein, the 3D5 antigen, precipitate the translation product of chick GAP-43 cDNA, and the 3D5 antigen is also detected by antisera against synthetic peptides from the known amino acid sequence of rat GAP-43. The chick protein and the rat GAP-43 are biochemically similar proteins that both serve as major targets of phosphorylation by endogenous protein kinase C. The detergent-resistant complex in which GAP-43 is found also contains actin (approximately 5% of the total protein) and a neurone-specific cell surface glycoprotein. We suggest that the membrane skeleton of neurones may be a primary site of action of GAP-43.
The present study examined the effect of codeine, a centrally acting opiate, on the respiratory sensations elicited in normal subjects by breathing to exhaustion against externally applied inspiratory threshold loads. Subjects were tested on two separate days following the double-blind, randomized administration of either placebo or codeine (90 mg). The intensity of the sensations of effort and discomfort experienced during two loaded breathing trials (a "high" load that was 73% of the maximum inspiratory pressure (MIP) and a "low" load that was 63% of the MIP) was evaluated using category (Borg) scores on each day of study. To verify that the dosage of codeine administered was sufficient to produce analgesia, we also determined the effect of this dosage on the time that subjects could tolerate immersion of one hand in ice water. Codeine altered neither the perceived effort nor the sense of discomfort associated with breathing against external loads and had no appreciable effect on the time to exhaustion during loaded breathing trials. This dose of codeine did, however, increase the time that ice water immersion could be tolerated and reduced the rate at which the sense of discomfort increased over time during ice water trials. These results indicate that, provided the pressure-time index of respiratory muscle contraction remains constant, analgesic doses of codeine alter neither the sensations elicited by loaded breathing nor the total time that breathing against a fatiguing inspiratory load can be tolerated.
Asherman's syndrome is reviewed, and 27 cases treated by us in Iran, England, New Zealand and Australia over a 20-year period are analysed. Aetiological factors and treatment are discussed. In view of the high incidence of complications in subsequent pregnancies, the need for prevention is stressed. Although more common in some countries, it is, nevertheless, of world-wide distribution and, unless looked for, will be missed.