Onset of changes in phospholipid fatty acid composition and prostaglandin synthesis following dietary manipulation with n-6 and n-3 fatty acids in the rat

A synthetic diet preparation supplemented with 10% by weight of either safflower oil, hydrogenated coconut oil containing 3% safflower oil, or 'max EPA' fish oil was fed to rats over a 8-week period. Serial measurements of serum fatty acids, serum thromboxane B2 and urinary prostaglandin excretion were taken during the treatment period to assess the rate of change in fatty acid composition and prostaglandin synthesis following dietary manipulation. There was no significant change in weight gain between the dietary groups during the treatment period. Significant changes in serum fatty acids occurred within 48 h of treatment, with the 'max EPA' oil group having arachidonic acid levels reduced by 23% (P less than 0.01) compared to the coconut oil group. Conversely, rats fed safflower oil had an 18% enhancement of arachidonic acid during the same time period. Whole blood synthesis of thromboxane B2 was significantly depressed (P less than 0.01) after 48 h in rats fed 'max EPA' oil compared to the safflower oil or coconut oil groups. This suppression reached a maximum of 65% (P less than 0.001) after 7 days of dietary 'max EPA' oil treatment. The safflower oil and coconut oil-fed groups showed the same levels of serum thromboxane B2 production over the treatment period. Urinary excretion of both 6-ketoprostaglandin F1 alpha and prostaglandin E2 varied significantly (P less than 0.01) between the groups after 7 days of dietary treatment. Rats fed 'max EPA' oil had depressed urinary prostanoid excretion compared to the safflower and coconut oil groups which remained very similar to each other. After the 8-week treatment period rats were killed and the phospholipid fatty acid composition and prostaglandin-generating capacity of platelets, aorta and renal tissue was examined. Prostanoid production by kidney cortex and medulla and segments of aorta was consistently suppressed in rats fed 'max EPA' oil. These observations correlated well with changes in the phospholipid fatty acid profiles in these tissues. This study shows rapid changes in serum fatty acids and thromboxane B2 generation following dietary manipulation, while changes in urinary excretion or prostanoid metabolites occur only after a longer time period.     

Emotional and behavioral reactions to facially deformed patients before and after craniofacial surgery

The present experiment investigated whether observers' emotional and behavioral reactions to facially deformed patients could be substantially improved by surgical procedures conducted by well-trained specialists in an experienced multidisciplinary team. Also investigated was the hypothesis that emotional states mediate the effects of physical attractiveness and facial deformity on social interaction. Twenty patients between the ages of 3 months and 17 years were randomly selected from over 2000 patients' files of Kenneth E. Salyer of Dallas, Texas. Patient diagnoses included facial clefts, hypertelorism, Treacher Collins syndrome, and craniofacial dysostoses (Crouzon's and Apert's syndromes). Rigorously standardized photographs of patients taken before and after surgery were shown to 22 "naive" raters ranging in age from 18 to 54 years. Raters were asked to predict their emotional and behavioral responses to the patients. These ratings indicated that observers' behavioral reactions to facially deformed children and adolescents would be more positive following craniofacial surgery. Similarly, the ratings indicated that observers' emotional reactions to these patients would be more positive following surgery. The results are discussed in terms of current sociopsychologic theoretical models for the effects of attractiveness on social interaction. A new model is presented that implicates induced emotional states as a mediating process in explaining the effects of attractiveness and facial deformity on the quality of social interactions. Limitations of the current investigation and directions for future research are also discussed.     

Effect of luteinizing hormone releasing hormone (LHRH) and [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide on alpha-subunit and LH beta messenger ribonucleic acid levels in rat anterior pituitary cells in culture

The effect of incubation with LHRH and its agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide has been measured on the concentrations of mRNAs for the common alpha-subunit of glycoprotein hormones and beta-LH in rat anterior pituitary cells in primary culture. After incubation, total RNA was analyzed by Northern blot or dot blot hybridization with alpha- and LH beta 32P-labeled cRNA probes and mRNA levels were quantified by autoradiography. Short-term treatment (4-6 h) of pituitary cells with 100 nM LHRH led to a marked stimulation of LH release but no effect was observed on alpha-subunit or LH beta mRNA levels. Longer (24-72 h) incubation periods with LHRH led to complete desensitization of the LH response to the neurohormone and induced 2- to 3-fold increases in alpha-mRNA cell content while LH beta mRNA levels remained unchanged. Maximal induction of alpha mRNA accumulation was observed with an LHRH concentration as low as 0.1 nM. Incubation with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide for 24-72 h also increased alpha mRNA but did not modify LH-beta mRNA levels. It is concluded that long-term exposure of anterior pituitary cells to LHRH or to an LHRH agonist positively regulates alpha-subunit gene expression in the absence of change in LH beta mRNA levels. This observation can provide an explanation for the high plasma levels of free alpha-subunits found in patients treated chronically with LHRH agonists.     

Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.     

Effect of alpha-adrenergic stimulation on prostacyclin and renin release in the rat kidney

The relationship between renin secretion and PGI2 production, in response to intrarenal infusion of norepinephrine, was examined in the isolated perfused rat kidney. Infusion of norepinephrine in a dose which caused substantial vasoconstriction (100 ng/min), markedly increased urinary excretion of 6-keto PGF1 alpha, the stable derivative of PGI2, without significantly altering renin secretion measured in the effluent perfusate. No change in urinary 6-keto PGF1 alpha occurred when vasoconstriction was prevented by infusing the alpha-adrenoceptor blocking drug phenoxybenzamine (2 x 10(3) ng/min) alongside norepinephrine (100 ng/min). However, under these conditions there was marked stimulation of renin secretion which, as has been demonstrated previously, is mediated by a beta-adrenoceptor. There were significant increases in urine flow rates during both vasoconstrictor and non-vasoconstrictor infusions. These findings clearly indicate that in the rat kidney prostacyclin production and renin release in response to norepinephrine are dissociated.     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay

We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an ED50 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, beta-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 ng/mg tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.