Detection of a C-insertion polymorphism within the human tumor necrosis factor alpha (TNFA) gene

We have identified a C-insertion polymorphism in the 5'UTR of the first exon of the human tumor necrosis factor alpha (TNFA) gene. TNFA is a cytokine that plays an important role in the inflammatory response.     

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

Soluble Leukocyte-Associated Ig-Like Receptor-1 in Amniotic Fluid Is of Fetal Origin and Positively Associates with Lung Compliance

The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance suggests that amniotic fluid sLAIR-1 may be useful as a novel independent marker of neonatal lung maturation.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

Receptor-mediated ADP-ribosylation of a phospholipase C-stimulating G protein

In membranes of myeloid differentiated HL 60 cells, the chemotactic peptide FMLP stimulates phospholipase C via a pertussis toxin-sensitive G protein. FMLP markedly stimulates the cholera toxin-dependent ADP-ribosylation of a 40 kDa protein in these membranes. This effect of FMLP is inhibited by GTP and GTP[S], and is almost completely abolished in membranes of pertussis toxin-pretreated HL 60 cells. Treatment of HL 60 membranes with cholera toxin and NAD markedly inhibits FMLP-stimulated high affinity GTPase. These results suggest that a 40 kDa G protein sensitive to both pertussis and cholera toxin functionally interacts with the formyl peptide receptor of HL 60 cells and, thus, very likely is the G protein that stimulates phospholipase C in this system.     

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

Dietary zinc deficiency has no effect on auditory brainstem responses in the rat

Zinc has been shown to effect--in vitro--a number of processes associated with neurotransmission. We have tested whether the rate of impulse conduction--in vivo--as measured from the latencies of auditory brainstem responses (ABR), is influenced by dietary zinc deficiency in the rat. Dietary zinc deficiency for up to 26 wk had no effect on the wave I-IV interval compared to zinc-adequate fed animals. The results are discussed in relation to the observed constancy of brain overall and extracellular fluid zinc concentrations under conditions of dietary zinc deficiency.     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.     

Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.