Neuropathy Target Esterase: Rates of Turnover In Vivo Following Covalent Inhibition with Phenyl Di-n-Pentylphosphinate

Phenyl di-n-pentylphosphinate is a reasonably stable easily synthesized inhibitor of neuropathy target esterase (NTE) with low anticholinesterase activity. Like phenylmethylsulphonyl fluoride it protects hens against neuropathic effects of compounds such as diisopropylphosphorofluoridate. At intervals up to 15 days after dosing hens (10 mg/kg s.c. to inhibit 90% NTE) assays were made of catalytically active and of phosphinylated NTE in autopsy tissue. The sum of these components was always within the range of catalytic activity in undosed controls. However, the half-life of reappearance of active NTE was 2.07 days +/- 0.13 (SD, n = 6) for brain and 3.62 days +/- 0.23 (SD, n = 6) for spinal cord--shorter than after dosing with phenylmethylsulphonyl fluoride. It is proposed that: (1) The physiological turnover mechanism cannot distinguish between catalytically active and di-n-pentylphosphinylated NTE although initiation of organophosphate-induced delayed polyneuropathy might involve recognition of aged di-alkyl-phosphorylated NTE as "foreign". (2) The short half-lives indicate a slow spontaneous dephosphinylation of inhibited NTE occurs in vivo as well as de novo synthesis. The difference in half-lives for brain and spinal cord NTE may be due to different rates of synthesis de novo or (more likely) to different rates of spontaneous reactivation of the inhibited NTE in the two tissues.     

Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate

In the cultured acute lymphoblastic leukemic (ALL) cell line, clones of sensitive cells are killed by receptor-occupying concentrations of glucocorticoids. In addition, several types of resistance have been identified. The types of resistance are r- (glucocorticoid binding site loss), ract/l (activation labile receptors) and r+ly- (defective lysis mechanism). The two types of receptor mutants have been examined for the presence and expression of the glucocorticoid receptor (GR) gene. Southern blot analysis, using a full-length cDNA probe for human GR, shows that the gene in both is grossly intact. Examination of the expression of the gene by Northern blots reveals the presence of normal, 7-kb message in both types of receptor mutants, though in amounts somewhat reduced from wild-type. This report focuses on the activation labile mutants. Since characterization of these mutants suggests that they can bind ligand but not retain it during activation, we hypothesized that they would respond normally to a ligand that could not be lost during activation. This seems to be the case. When the covalent affinity ligand dexamethasone mesylate, itself a partial glucocorticoid agonist/antagonist, is used, the ract/l cells are killed to an extent corresponding to that evoked by a sub-optimal concentration of the full agonist dexamethasone. We conclude: (1) that the ract/l receptors can function to kill cells if provided a ligand that they do not lose during activation; (2) that the partial agonist activity of dexamethasone mesylate for cell killing is not due to release of a small amount of free dexamethasone; (3) that the poor agonist activity of dexamethasone mesylate receptor complexes suggests that the role of steroid is strictly to participate in conversion of the receptor to its DNA binding form, after which presence of the steroid actually interferes with proper receptor action.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Calcitonin gene-related peptide in human fetal lung and in neonatal lung disease

The ontogeny of calcitonin gene-related peptide immunoreactivity (CGRP-IR) was evaluated immunohistochemically in 67 human fetal or newborn lungs previously analyzed for calcitonin immunoreactivity (CT-IR). CGRP-IR was present by 10 weeks of gestation in rare, solitary neuroendocrine (NE) cells of developing conducting airways in two of eight first-trimester lungs. During the second trimester, cells with CGRP-IR were found consistently (21/23 fetuses). However, the numbers of positively staining cells did not appear to increase in these fetuses or in third-trimester infants dying of non-pulmonary causes. The highest concentrations of CGRP-IR cells were seen in lungs of premature infants with advancing chronic lung disease associated with bronchopulmonary dysplasia (BPD). CGRP-IR was seen earlier in gestation and in greater numbers of NE cells than was calcitonin immunoreactivity (CT-IR) reported previously in these same fetal lungs (Lab Invest 52:52, 1985). Its presence paralleled that of CT-IR in postnatal chronic lung disease.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Receptor activation of G proteins

G proteins are a highly conserved family of membrane-associated proteins composed of alpha, beta, and gamma subunits. The alpha subunit, which is unique for each G protein, binds GDP or GTP. Receptors such as those for beta- and alpha-adrenergic catecholamines, muscarinic agonists, and the retinal photoreceptor rhodopsin, catalyze the exchange of GDP for GTP binding to the alpha subunit of a specific G protein. G alpha.GTP regulates appropriate effector enzymes such as adenylyl cyclase or the cyclic GMP phosphodiesterase. The beta gamma-subunit complex of G proteins is required for efficient receptor-catalyzed alpha subunit guanine nucleotide exchange and also functions as an attenuator of alpha subunit activation of effector enzymes. Recent elucidation of both receptor and G protein primary sequence has allowed structural predictions and new experimental approaches to study the mechanism of receptor-catalyzed G protein regulation of specific effector systems and the control of cell function including metabolism, secretion, and growth.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Subcutaneous narcotic infusions for cancer pain: treatment outcome and guidelines for use.

OBJECTIVE: To provide guidelines for the institution and maintenance of a continuous subcutaneous narcotic infusion program for cancer patients with chronic pain through an analysis of the narcotic requirements and treatment outcomes of patients who underwent such therapy and a comparison of the costs of two commonly used infusion systems. DESIGN: Retrospective study. SETTING: Tertiary care facilities and patients'' homes. PATIENTS: Of 481 patients seen in consultation for cancer pain between July 1987 and April 1990, 60 (12%) met the eligibility criteria (i.e., standard medical management had failed, and they had adequate supervision at home). INTERVENTION: Continuous subcutaneous infusion with hydromorphone hydrochloride or morphine started on an inpatient basis and continued at home whenever possible. OUTCOME MEASURES: Patient selectivity, narcotic dosing requirements, discharge rate, patient preference for analgesic regimen, side effects, complications and cost-effectiveness. RESULTS: The mean initial maintenance infusion dose after dose titration was almost three times higher than the dose required before infusion (hydromorphone or equivalent 6.2 v. 2.1 mg/h). Eighteen patients died, and the remaining 42 were discharged home for a mean of 94.4 (standard deviation 128.3) days (extremes 12 and 741 days). The mean maximum infusion rate was 24.1 mg/h (extremes 0.5 and 180 mg/h). All but one of the patients preferred the infusion system to their previous oral analgesic regimen. Despite major dose escalations nausea and vomiting were well controlled in all cases. Twelve patients (20%) experienced serious systemic toxic effects or complications; six became encephalopathic, which necessitated dose reduction, five had a subcutaneous infection necessitating antibiotic treatment, and one had respiratory depression. The programmable computerized infusion pump was found to be more cost-effective than the disposable infusion device after a break-even point of 8 months. CONCLUSIONS: Continuous subcutaneous infusion of opioid drugs with the use of a portable programmable pump is safe and effective in selected patients who have failed to respond to standard medical treatment of their cancer pain. Dose titration may require rapid dose escalation, but this is usually well tolerated. For most communities embarking on such a program a programmable infusion system will be more cost-effective than a disposable system.