Production of soluble surface glycoprotein, GP70, of the Mason-Pfizer monkey virus in COS cells is dependent on the presence of regions of the transmembrane domain.
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Animals↗
Biomedical subjects
J M Allen
Publications and source records attributed to J M Allen.
Beta-adrenergic receptor downregulation and recovery in heart and lung in the Fischer 344 rat: effects of aging and correlation with mRNA levels.
Using indirect methods to downregulate beta-adrenergic receptor (BAR) density, several investigators have observed an aging-related delay in the recovery of BAR density after downregulation and suggested this finding may in part explain the decreased beta-adrenergic responsiveness associated with aging. We downregulated BAR density in male Fischer 344 rats ages 3, 12, and 24 months using the direct BAR agonist, metaproterenol. Lung and heart BAR density and BAR mRNA levels were measured daily for 5 days during recovery. Heart BAR density was downregulated significantly more in the 24-month compared to the 3-month-old animals (ANOVA p < .05). The rate of recovery of heart BAR density was greater in the 24-month compared to the 3-month-old animals (ANOVA, p = .05). Lung BAR density showed no significant age-related differences at baseline, after downregulation, or during recovery. There was no significant change in lung or heart BAR mRNA levels observed in association with downregulation of BAR density. The previously documented decrease in myocardial beta-adrenergic responsiveness with aging in this animal appears to not be related to delayed or incomplete recovery of BAR density after agonist-induced downregulation, but may be associated with greater initial downregulation of heart BAR density.
Aging↗
Detection of reverse transcriptase in culture medium for mammary tumour cell lines: a comparison of an established radio-labelling technique and a contemporary non-isotopic technique.
Classically, radio-label techniques have been employed to analyse biological samples for reverse transcriptase (RT) activity. More recently, however, non-isotopic kits have been developed for retroviral quantification. Nevertheless, until the present investigation it has not been known if these contemporary methods are more sensitive at detecting reverse transcriptase activity. In our study, a non-isotopic ELISA method was shown to be considerably more sensitive than the radio-label technique at detecting reverse transcriptase in growth medium used to culture the murine breast cancer cell line GR/A. Using the ELISA, less reverse transcriptase activity was demonstrated in growth medium from human mammary adenocarcinoma MCF-7 cells than the murine source. This ELISA did not detect reverse transcriptase activity from a pure source of Moloney murine leukaemia virus. In light of this, the broad applicability of this ELISA for reverse transcriptase from different viral sources must be investigated before it can be used to monitor biological supernatants for the presence of retroviruses.
Animals↗
Binding of monomeric immunoglobulin G triggers Fc gamma RI-mediated endocytosis.
Cross-linking of leukocyte Fc receptors specific for IgG (Fc gamma Rs) by multivalent IgG complexes triggers a wide range of immune functions. Many of these responses can also be stimulated in vitro using anti-Fc gamma R monoclonal antibody-containing complexes. This observation has suggested that cross-linking is the key event and that binding of IgG, which in itself does not elicit a response, is functionally passive. However, in this study we show that binding of monomeric IgG to the human high affinity receptor, Fc gamma RI, is itself sufficient to permit the receptor to enter an internalization-recycling pathway, which has a small intracellular pool. Unoccupied Fc gamma RI is not internalized and recycled in this manner. This finding may be explained by the previous observation that there is a physical association between Fc gamma RI and the cytoskeletal component, actin-binding protein (non-muscle filamin; ABP-280), which is disrupted upon IgG binding. Thus, in the absence of IgG, Fc gamma RI may be physically excluded from the endocytic pathway by tethering to the cytoskeleton. The role of cross-linking is to divert Fc gamma RI-IgG complexes from the recycling pathway, causing their retention and subsequent degradation within the cell. In contrast to Fc gamma RII-mediated endocytosis, intracellular accumulation of cross-linked Fc gamma RI-IgG complexes is not sensitive to inhibition by genistein, suggesting that the process is independent of tyrosine kinase activity.
Antigen-Antibody Complex↗
A convenient method for the construction and expression of GPI-anchored proteins.
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Amino Acid Sequence↗
Convergent pathways for steroid hormone- and neurotransmitter-induced rat sexual behavior.
Estrogen and progesterone modulate gene expression in rodents by activation of intracellular receptors in the hypothalamus, which regulate neuronal networks that control female sexual behavior. However, the neurotransmitter dopamine has been shown to activate certain steroid receptors in a ligand-independent manner. A dopamine receptor stimulant and a D1 receptor agonist, but not a D2 receptor agonist, mimicked the effects of progesterone in facilitating sexual behavior in female rats. The facilitory effect of the neurotransmitter was blocked by progesterone receptor antagonists, a D1 receptor antagonist, or antisense oligonucleotides to the progesterone receptor. The results suggest that in rodents neurotransmitters may regulate in vivo gene expression and behavior by means of cross-talk with steroid receptors in the brain.
Adrenal Glands↗
Nitric oxide mediates sexual behavior in female rats.
Nitric oxide (NO), an active free radical formed during the conversion of arginine to citrulline by the enzyme NO synthase (NOS), mediates vasorelaxation, cytotoxicity, and neurotransmission. Neurons containing NOS (NOergic) are located in the hypothalamus. These NOergic neurons control the release of several hypothalamic peptides. Release of NO from these NOergic neurons stimulates pulsatile release of luteinizing hormone-releasing hormone (LHRH) in vivo and LHRH release in vitro. LHRH not only induces LH release, which induces ovulation, but also facilitates female sexual behavior. Sexual behavior can be induced reliably in estrogen-primed ovariectomized female rats by progesterone (P). This behavior consists of proceptive behavior to attract the male and the assumption of a clear characteristic posture, lordosis, when mounted by the male. To ascertain the role of NO in the control of sexual behavior in female rats, an inhibitor of NOS, NG-monomethyl-L-arginine was microinjected into the third cerebral ventricle (3V) of conscious, ovariectomized, estrogen-primed rats with indwelling cannulae. NG-Monomethyl-L-arginine (10-1000 micrograms) prevented P-facilitated lordosis when administered intracerebroventricularly into the 3V, 20 min prior to the 3V injection of P. NG-Monomethyl-D-arginine, which does not inhibit NOS, did not inhibit lordosis under the same experimental conditions. Microinjection into the 3V of sodium nitroprusside (SNP), which spontaneously releases NO, facilitated lordosis in estrogen-primed rats in the absence of P. The facilitation of lordosis induced by either P or SNP was prevented by intracerebroventricular injection of hemoglobin, which binds NO. Lordosis facilitated by P or SNP was blocked by injection of LHRH antiserum into the 3V. The results are interpreted to mean that the P-facilitated lordosis response is mediated by LHRH release. Furthermore, since NO release from SNP also facilitates lordosis in the absence of P and this response could be blocked by LHRH antiserum, we conclude that P brings about the release of NO, which stimulates LHRH release that facilitates lordosis. Thus, the results indicate that NO induces LHRH release and that LHRH then plays a crucial role in mediation of sexual behavior in the female rats.
Amino Acid Oxidoreductases↗
Low intensity laser irradiation inhibits tritiated thymidine incorporation in the hemopoietic cell lines HL-60 and U937.
The purpose of this study was to determine the effect of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on tritiated thymidine incorporation in two hemopoietic cell lines, HL-60 and U937. Cells were suspended at a concentration of 1 x 10(6)/ml in their respective serum-free media and irradiated at energy densities from 1.0 to 11.5 J/cm2. Twenty-four hours after irradiation the cells were assayed for their ability to incorporate tritiated thymidine (3H-TdR) in comparison with nonirradiated cells. Analysis by two-way analysis of variance (ANOVA) for unrelated groups showed that laser irradiation at all energy densities > or = 5.8 J/cm2 produced a significant decrease in 3H-TdR incorporation (P < 0.05) into HL-60 cells. In U937 cells, irradiation at energy densities of 5.8, 7.2, and 11.5 J/cm2 caused a similar reduction in 3H-TdR incorporation (P < 0.01), although not at 8.6 and 9.6 J/cm2. The temperature of each cell suspension was recorded both during and immediately postirradiation, and no significant thermal changes were observed. These findings demonstrate a direct photobiological effect of laser irradiation on these two cell lines. The precise mechanism for this effect is unknown but may have significance in understanding the biological action of laser's known therapeutic effectiveness in promoting wound repair.
Cell Death↗
Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve: relevance of radiant exposure.
The effects of low intensity near-infrared laser radiation (830 nm; 1.5-12 J/cm2; continuous wave) on peripheral neurophysiology and skin temperature were investigated using antidromic conduction studies in the human median nerve in vivo. Healthy human volunteers (n = 80) were recruited and randomly allocated to one of two control (n = 30) or five laser groups (1.5, 3.0, 6.0, 9.0, and 12 J/cm2; n = 10 per group). Analysis of variance (ANOVA) in negative peak latency difference scores (NPLDs) and skin temperature over a 20 min period post-irradiation demonstrated a significant decrease in skin temperature following irradiation at the lowest radiant exposure (1.5 J/cm2) coupled with a significant increase in NPLDs (P < 0.05). While no such (significant) changes were found in the other laser groups, it was found that observed changes (increases and decreases) in NPLDs were inversely related to changes in skin temperature. These findings demonstrate measurable peripheral neurophysiological effects of laser radiation concomitant with changes in skin temperature; however, further studies are indicated to establish the precise relationship between the observed effects on skin temperature and nerve conduction.
Adolescent↗
Role of protein kinase C in mediating NGF effect on neuropeptide Y expression in PC12 cells.
Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous systems. Treatment with Nerve Growth Factor (NGF) induces an increase in NPY mRNA in PC12 cells, a rat pheochromocytoma cell line extensively used as a model of neuronal differentiation. Stimulators of both cAMP and calcium-phospholipid dependent protein kinases (PKA and PKC respectively) increase NPY mRNA levels in a similar way to NGF. Nevertheless, H-89, a specific inhibitor of PKA failed to block NGF stimulated NPY mRNA accumulation. Furthermore, direct measurement of PKA activity in cell extracts showed no increase following NGF, in contrast to forskolin. H7, an inhibitor of both PKC and PKA systems completely abolished the NGF induced increase in NPY mRNA, suggesting that PKC is necessary for NGF induction of the NPY gene. NGF also increased PKC activity in cell extracts in a similar way to phorbol myristate acetate (PMA). Use of a reporter function, chloramphenicol acetyl transferase, controlled by 700 base pairs of the 5' flanking region of the NPY gene demonstrated that NGF and phorbol ester stimulated transcription of the NPY gene. This stimulation could be blocked by pre-incubating PC12 cells with calphostin C, a specific inhibitor of PKC. Our results indicate that NGF induces NPY gene expression via activation of PKC system. Although an increase in adenylate cyclase activity affects the expression of the NPY gene, activation of PKA appears not to be involved in mediating the NGF effects.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Cytosolic calcium elevation in response to Fc receptor cross-linking in undifferentiated and differentiated U937 cells.
We have used the calcium indicator, Fura-2, to investigate cytosolic calcium responses to cross-linking of monomeric IgG-occupied surface Fc gamma receptors (Fc gamma R), using populations of the human monocyte-like cell line, U937. The magnitude and duration of the calcium response observed, and the relative contribution to the response of internal stores and external calcium, are found to depend on the state of differentiation of these cells. Initial release of calcium from stores following Fc gamma R cross-linking is enhanced by prior treatment of U937 cells with both interferon-gamma, and, to a lesser extent, with dibutyryl cAMP. A large and prolonged entry of external calcium is observed in dibutyryl cAMP treated cells; this may be due to direct regulation of calcium channels by the low affinity receptor, Fc gamma RII (whose expression is up-regulated in these cells), since the smaller and more transient entry observed in interferon-gamma treated cells, (where the high affinity receptor, Fc gamma RI, is up-regulated) argues against a common pathway of store-mediated calcium entry.
Bucladesine↗
Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo.
The current investigation, for which ethical permission was obtained, was designed to assess the putative neurophysiological effects of low intensity (9.6 J/cm2) laser (830 nm) irradiation upon conduction latencies in the human median nerve in vivo. Antidromic nerve conduction studies were performed on the non-dominant arms of healthy human volunteers (n = 51), who were each assigned to one of a variety of laser, placebo or control groups under randomized, blinded conditions. Analysis of negative peak latency differences using analysis of variance showed small (approximately 0.4 ms) but significant increases in latencies as a result of direct laser irradiation, and, where two-site recording was used, distal to the site of irradiation. No such differences were seen in the other experimental groups. These results show that laser irradiation applied to intact skin at the parameters used here may produce a direct, localized effect upon conduction in underlying nerves.
Adult↗
Inhibition of rat sexual behavior by antisense oligonucleotides to the progesterone receptor.
To test further the idea that sexual behavior in rodents is mediated via the progesterone receptor (PR) in the ventromedial nucleus of the hypothalamus, antisense and sense oligonucleotides to progesterone receptor were administered intracerebroventricularly into the third cerebral ventricle of ovariectomized estrogen-primed animals. Progesterone-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides, with proceptive and receptive responses being minimal or completely suppressed. Sexual behavior was not altered by control sense oligonucleotides. In vitro binding assays of the cytosol progesterone receptors demonstrated a 52.2% reduction of PRs in the hypothalamus of animals that received antisense oligonucleotides, suggesting a reduction in PR synthesis. These data suggest that a threshold level of estrogen-induced hypothalamic PR is critical in the regulation of progesterone-facilitated sexual behavior in female rats.
Animals↗
Human high-affinity Fc IgG receptor (Fc gamma RI)-mediated phagocytosis and pinocytosis in COS cells.
The high-affinity receptor (Fc gamma RI) for the constant (Fc) portion of immunoglobulin G (IgG) is one of three Fc IgG receptor classes (Fc gamma Rs) found on mononuclear phagocytes. The functional specialization of each of the Fc gamma R classes is not well understood. Previous studies utilizing anti-Fc gamma R monoclonal antibodies (mAbs) as opsonins suggest that Fc gamma RI, like the other Fc gamma Rs expressed by macrophages, is able to mediate phagocytosis. The ability of Fc gamma RI to mediate pinocytosis, however, had not been certain, since it binds, but does not mediate, internalization of monomeric IgG in the monocytoid U937 cells. We studied Fc gamma RI-mediated internalization by introducing it into the Fc gamma R-negative fibroblastic COS cells. We found, using electron microscopy and fluorescence microscopy, that COS cells expressing Fc gamma RI are able to phagocytose IgG-coated zymosan particles and sheep red blood cells (SRBC), as well as pinocytose cross-linked IgG. There was no intracellular accumulation of monomeric IgG. Chimeric receptors which retain the extracellular domains of Fc gamma RI but lack the entire wild-type transmembrane and intracellular regions of the receptor mediated both phagocytosis and pinocytosis with equal or increased efficiency when compared to the wild-type receptor. Control COS cells transfected with CD2 rosetted, but did not phagocytose, SRBC. Attachment of phagocytic targets to COS cells is therefore not sufficient for phagocytosis. Taken together, this suggests that the extracellular domain of Fc gamma RI is sufficient for it to mediate phagocytosis and pinocytosis in this system.
Amino Acid Sequence↗
Vasoactive intestinal peptide stimulates neuropeptide Y gene expression and causes neurite extension in PC12 cells through independent mechanisms.
Vasoactive intestinal peptide (VIP) is widely recognized as a regulator of tyrosine hydroxylase via a mechanism of trans-synaptic activation. Subsets of adrenal medullary cells and postganglionic sympathetic nerves coexpress the peptide neurotransmitter neuropeptide Y (NPY) with catecholamines. Using PC12 cells transiently expressing a fusion gene in which the bacterial enzyme chloramphenicol acetyltransferase (CAT) is under the control of 700 base pairs of the 5' flanking region of the NPY gene, we have studied the role of VIP and the related peptide pituitary adenylate cyclase activating peptide (PACAP) in regulating NPY gene transcription. Both VIP and PACAP stimulated expression of the NPY gene through activation of cAMP-dependent protein kinase. PACAP was 1000-fold more potent in eliciting this response compared to VIP and activity resided in its N-terminal 27 amino acids. Both VIP and PACAP caused a subpopulation (approximately 50%) of PC12 cells to undergo profound morphological changes in that the cells extended long, slender neurites with prominent growth cones. This change in morphology was unaffected by preincubating cells with inhibitors of either cAMP-dependent protein kinase or calcium/phospholipid-dependent protein kinase. A trophic role for either VIP or PACAP in regulating sympathetic nerve function is proposed.
Animals↗
Unlicensed practice.
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Legislation, Drug↗
Aqueous-phase photochemical formation of peroxides in authentic cloud and fog waters.
Gas-to-drop partitioning of hydrogen peroxide and its precursor, the hydroperoxyl radical (HO2.), has been considered the predominant or sole source of hydrogen peroxide in atmospheric water drops. However, atmospheric water can absorb solar ultraviolet radiation, which initiates the photoformation of peroxides (primarily hydrogen peroxide). Measurements of peroxide photoformation rates in authentic atmospheric water samples demonstrate that aqueous-phase photochemical reactions are a significant, and in some cases dominant, source of hydrogen peroxide to cloud and fog drops. This additional source could significantly change the current understanding, and hence, the models, of sulfuric acid deposition because hydrogen peroxide is the limiting reagent in the dominant pathway for the oxidation of sulfur dioxide to sulfuric acid in the troposphere over eastern North America.
Atmosphere↗
Identification of a neuron-specific promoter of human aromatic L-amino acid decarboxylase gene.
We have cloned the 5' region of human aromatic L-amino acid decarboxylase (AADC) gene in a cosmid and an overlapping lambda clone, and sequenced the first five exons. A 61 base pair (bp) non-coding, first exon containing for the 5' end of a human pheochromocytoma AADC cDNA was localized 16 kb upstream of exon 2, in which translation is initiated. The transcription start site was localized by RNAse mapping, primer extension and reverse transcription-PCR. The non-conventional cap site was preceded by a modified TATA box at position -29. A strong promoter was characterized in the 560 bp region upstream of the cap site by linkage to the reporter gene LacZ, and transfection in human neuroblastoma SK-N-BE and SK-N-BE-K2 cells. Using a series of constructs bearing a varying length of 5' flanking region, three positive regulatory elements have been localized in the -560 to -394, -244 to -200 and -147 to -1 regions. Negative regulatory elements were localized in the -9000 to -560 and -394 to -316 regions. Surprisingly, constructs comprising all or the major part of intron 1 were inactive, suggesting the presence of a silencer in the first intron, or incorrect splicing events. The construct containing 560 bp of 5' flanking sequence did not express in human cholinergic neuroepithelioma cells MC-I-XC, and in three non-neuronal cell lines which displayed high AADC activities: human pancreatic carcinoma cells AsPC-1, rat insulinoma cells RINm5F and mouse anterior pituitary cells AtT20. These data suggest that we have identified a neuron-specific AADC promoter. An extensive search for a second promoter responsible for AADC gene expression in non-neuronal cells only gave negative results.
Amino Acid Sequence↗