Localization of chemotactic peptide receptors on rabbit neutrophils

The chemotaxis of blood leukocytes is initiated by the binding of a chemoattractant to specific receptors on the leukocyte cell surface [1, 2]. Although a great deal is known about the biochemical and morphological events accompanying chemotactic activation [3-9], there is very little morphological information about the chemoattractant receptors themselves. This latter information is needed so that we may understand the mechanism by which these inflammatory cells detect and respond to chemical gradients. One class of chemotactic factors extensively used to characterize the complex behavioral responses following leukocyte activation are the synthetic formylmethionyl peptides. These peptides, now known to be the analogs of the naturally occurring N-terminal peptides produced by bacteria [10], are released into culture medium and are believed to be responsible, at least in part, for the accumulation of leukocytes at the sites of bacterial infection [1, 2]. We have localized the receptors for the chemotactic hexapeptide N-formylnorleucyl-leucyl-phenylalanine-norleucyl-[125I]tyrosyl-lysine [N-fNle---Leu---Phe---Nle---[125I]Tyr---Lys] on whole rabbit peritoneal neutrophils (PMN) using light microscope autoradiography. By this method, the inherent formylpeptide receptor distribution on cells incubated at 4 [deg]C appears to be uniform over the surface of both rounded and structurally polarized PMN. Following a short 37 [deg]C incubation, cells retain a large proportion of labelled hexapeptide at or near the cell surface and, in addition, polarized PMN redistribute the hexapeptide anteriorly away from the cell uropod.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24692/1/0000111.pd

Nicotinamide Phosphoribosyltransferase/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of “NAMPT-mediated systemic NAD biosynthesis.” Our study would advance current understanding of visfatin physiology

Improving communication and recall of information in paediatric diabetes consultations: a qualitative study of parents' experiences and views

Background Parents of non-adolescent children with type 1 diabetes are responsible for most of their child’s diabetes management tasks. Consultations are used to provide diabetes education, review clinical progress and promote diabetes management tasks. This study explored parents’ experiences of, and views about, their child’s diabetes consultations. The objective was to identify ways in which consultations could be improved to aid communication, understanding and knowledge retention. Methods In-depth interviews with 54 parents of children (aged ≤12 years) with type 1 diabetes. Data were analysed using an inductive thematic approach. Results Parents’ accounts revealed structural and contextual factors which could hinder effective communication and knowledge acquisition during consultations. Most reported feeling anxious going into consultations and worrying about being reprimanded by health professionals if their child’s glycaemic control had not improved. As a consequence, many parents highlighted problems concentrating and assimilating information during consultations. In extreme cases, worries about being reprimanded led parents to omit or fabricate information when discussing their child’s treatment or even to their cancelling appointments. Many parents described wanting opportunities to speak to health professionals alone because young children could be distracting and/or they did not want to raise distressing issues in front of their child. Parents described the benefits of receiving clinical advice from health professionals familiar with their family circumstances and disliking attending busy clinics and seeing different health professionals on each occasion. Parents also highlighted the benefits of receiving treatment recommendations in a written form after the consultation. Discussion and conclusions This study has highlighted unrecognised and undocumented aspects of the consultation which may result in parents leaving uncertain about the main issues discussed and with questions unanswered and support needs unaddressed. Structural and contextual changes to consultations are recommended to improve concentration, knowledge acquisition and retention. These include: sending letters/written summaries after consultations highlighting key decisions, providing opportunities for parents to consult health professionals without their child being present, encouraging parents to ask more questions during consultations, having procedures in place to promote continuity of care and providing parents with consistent and non-contradictory advice

Transforming growth factor beta activates protein kinase C in microvessels isolated from immature rat brain

We investigated the activation of protein kinase C in microvessels isolated from rat brain. We found that unstimulated kinase activity in microvessels from immature animals is soluble while that from adults is particulate. The tumor promoter, phorbol 12-myristate 13-acetate, and the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, caused the redistribution of protein kinase C activity to the membrane fraction in microvessels from immature rats. Exposure to transforming growth factor beta resulted in similar redistribution of kinase activity. To our knowledge, this is the first report of an effect of transforming growth factor beta on protein kinase C. The kinase activity in microvessels from adult animals was unaffected by exposure to these agonists. We suggest that protein kinase C activation promotes differentiation of the brain microvasculature. Transforming growth factor beta may mediate this process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27423/1/0000461.pd

Effects of protein kinase inhibitors 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and N-[2-guanidinoethyl]-5-isoquinolinesulfonamide hydrochloride (HA1004) on calcitriol-induced differentiation of HL-60 cells

HL-60 promyelocytic leukemia cells were induced to differentiate by 1,25-dihydroxyvitamin D3 (calcitriol) into mature monocytes. Differentiation was assessed by nitro blue tetrazolium dye reduction, nonspecific esterase activity, and DNA synthesis. Terminal differentiation of cultures induced by calcitriol (10 nM) was inhibited by 80% when cells were treated simultaneously with protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) (32 [mu]M) and N-[2-guanidinoethyl]-5-isoquionlinesulfonamide hydrochloride (HA1004) (320 [mu]M). The 50 for inhibition of calcitriol-induced differentiation was approximately 15 [mu]M for H-7 and 170 [mu]M for HA1004. The IC50 values for H-7 and HA1004 antagonism of calcitriol-induced differentiation are quantitatively and relatively correlated to their known action to inhibit protein kinase C activity. Treatment of cells with concentrations of 0-32 [mu]M H-7 or 0-320 [mu]M HA1004 alone did not affect cell growth, differentiation, or trypan blue exclusion. However, higher concentrations of H7 (> 32 [mu]M) and HA1004 (> 320 [mu]M) were found to be cytotoxic. The data presented suggest that calcitriol-induced differentiation is antagonized by inhibitors of protein kinase and are consistent with the hypothesis that kinase C activity is required for HL-60 cell differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27400/1/0000432.pd

Lead activates protein kinase C in immature rat brain microvessels

We investigated the effects of inorganic lead upon calcium-, phospholipid-dependent protein kinase (protein kinase C) in brain microvessels isolated from 6-day-old rat pups. We found that (a) in broken cell preparations, lead at micromolar concentrations activates this enzyme to an extent equivalent to that of micromolar calcium (10.3 +/- 1.3 and 9.2 +/- 1.6 pmol/mg/min, respectively) and (b) preincubation ofintact microvessels with lead results in a translocation of protein kinase C from the soluble to the particulate fraction. The cytosolic kinase activity stimulated by lead has the same requirements for diacylglycerol and phospholipid as the calcium-stimulated enzyme, suggesting that lead activates the kinase by mimicking calcium. The hypothesis that lead affects protein kinase C activity through a mechanism similar to that of calcium is supported by the similar time courses of substrate phosphorylation and dephosphorylation mediated by lead and calcium. When intact microvessels are preincubated with micromolar concentrations of lead, the translocation of protein kinase C occurs in a dose- and time-dependent manner. The relocalization is virtually complete at 0.1 [mu] lead and by 30 min of exposure. We propose that the sensitivity of protein kinase C to lead, described here in immature brain microvessels, makes this regulatory enzyme a potential mediator of lead toxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27099/1/0000091.pd

Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[ 3 H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[ 3 H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[ 3 H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[ 3 H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[ 3 H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38446/1/240270406_ftp.pd