David B. Millar

Studies on histidine-rich polypeptides from human parotid saliva.

Abstract The isolation of an histidine-rich polypeptide from human parotid saliva is described. This polypeptide, termed HRP-1, contains 17% histidine. HRP-1 is a neutral molecule and is a precursor of the cationic histidine-rich polypeptides found in saliva. Results of in vitro saliva incubations suggest that breakdown of HRP-1 is enzyme mediated. Degraded species are smaller in size, more cationic in charge, and higher in histidine content. It is concluded that the many histidine-rich polypeptides in saliva are not all individual gene products and are related by a proteolytic degradation.

Physicochemical characterization of generation 5 polyamidoamine dendrimers

The dispersity, size, and self-interaction of generation 5 polyamidoamine dendrimeric polymers with different terminal groups (surfaces) were characterized using several physicochemical techniques. Amino-surface dendrimers form oligomeric aggregates in aqueous solution, even in the presence of high salt concentrations (0.6M sodium phosphate). In contrast, the hydroxyl-surface polymer G5-OH behaves as a single homogeneous (or paucidisperse) species at low concentration. Measurements of density increment and the sedimentation and diffusion coefficients of G5-OH suggest a more swollen, porous structure than a globular protein of comparable mass. Measurements of the concentration dependence of sedimentation equilibrium of G5-OH in pH 7.2 phosphate buffer indicate the presence of significant electrostatic repulsion overlaid on weakly attractive interactions, leading to the formation of nonspecific aggregates at sufficiently high dendrimer concentration.

β-Endorphin's modulation of lymphocyte proliferation is dose, donor, and time dependent

We find that beta-endorphin (Bend) can have, positive, negative, or neutral dose-dependent effects on the mitogen-stimulated proliferation of human peripheral blood lymphocytes. The distribution of positive, negative, or neutral responses was nonrandom. In studies carried out over a year, we show that an individuals mitogen-stimulated lymphocyte proliferative response to Bend can change with time. We show that the inhibition induced by cortisol can be, in part, relieved by Bend. On the basis of our results and those of others in the field, we put forward a model that can qualitatively account for many of the observations we and other investigators have made.

β-Endorphin modulates T-cell intracellular calcium flux and c-myc expression via a potassium channel

To characterize the effect of beta-endorphin on T-lymphocyte activation, we examined its influence on membrane currents, intracellular calcium flux, and c-myc mRNA levels during mitogenic stimulation of Jurkat cells. While beta-endorphin weakly enhanced voltage-activated K+ currents of Jurkat cells by itself, it suppressed these currents in the presence of mitogen. Naloxone, by itself, also enhanced K+ current amplitude, but in the presence of mitogen partially reversed the suppressive effect of beta-endorphin. A 5-30 min exposure to beta-endorphin resulted in an increase in the rate of mitogen-stimulated intracellular calcium release and an increase in c-myc mRNA levels relative to controls. Longer exposure (1-2 h) to beta-endorphin retarded intracellular calcium release, and suppressed c-myc expression. The suppressive effects were reversed by naloxone and mimicked by the K+ channel blocker, tetraethylammonium ion. These data suggest that opiate receptors and K+ channels of Jurkat cells are functionally coupled in a way that modulates intracellular calcium release and c-myc expression - two key processes in T-cell mitogenesis.

Isolation and partial characterization of an histidine-rich polypeptide from parotid saliva of the monkey, Macaca nemestrina

1. 1. An histidine-rich polypeptide has been isolated from the parotid saliva of Macaca nemestrina by column Chromatographic methods and compared to a similar molecule from human parotid saliva. 2. 2. The fraction has been characterized by amino acid composition, molecular weight, electrophoretic behavior, ultra violet absorption and circular dichroic spectra. 3. 3. The pattern of appearance in saliva suggests the histidine-rich polypeptide is a secretory molecule. 4. 4. Additional histidine-rich polypeptides observed in saliva are probably degradation products of this fraction.

ß-endorphin modulates calcium channel activity in human neutrophils

10(-6) M n-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated Ca2+ flux in human neutrophils is characterized by a profile composed of two peaks of different amplitude and breadth. beta-Endorphin inhibited the magnitude and modulated the kinetics of the second peak in a manner which was dose-dependent and could reflect either negative cooperativity or heterogeneity of binding sites. The second peak arises from calcium channel activity since in the presence of nifedipine or EGTA it was not evident while the first peak was reduced about 24%. Similarly, at 15 degrees C, where we were unable to detect any channel activity, the first peak was diminished by 35% and beta-endorphin had no detectable effect on this peak. These results led us to conclude that the first peak is chiefly composed of Ca2+ recruited from cytosolic stores which are relatively insensitive to the above treatments and a smaller fraction of calcium originating in calcium channel activity. Hence, we reason that beta-endorphin modulates only the calcium ion flux arising from calcium channel function.

Quin-2 and Fura-2 measure calcium differently☆

Several fluorescent probes have been used in the past to monitor and to measure intracellular calcium and calcium fluxes. The most widely used of these probes are those developed by Tsien. We address the markedly different values obtained when comparing Quin-2 (the original probe) with Fura-2 (a second-generation probe). In most cases the values for intracellular calcium have been considered to be interchangeable for the different probes. Using several different hematopoietic cell lines we show that in no case do the two probes yield equivalent values.

Prostanglandin D2 modulates human neutrophil intracellular calcium flux and inhibits superoxide release via its ring carbonyl

We compared the effects of prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2) and various ketones on superoxide (OX) release by human neutrophils, which had been stimulated by N-formyl methionyl leucyl phenylalanine (FMLP). Our data suggested that the ring carbonyl of PGD2 is essential to its inhibitory effect on OX release, but the carbonyl group as a ketone, alone is not sufficient. Using the fluorescent Ca2+ probe, Fura-2AM, we found that PGD2 increased the rate of decline of FMLP stimulated intracellular free Ca2+ (Ca)i, but that PGF2 had no effect. cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. Furthermore, the ring carbonyl of PGD2 is crucial to its effect on OX as well as on (Ca)i.

The K channel blocker, tetraethylammonium, displaces beta-endorphin and naloxone from T-cell binding sites

Beta-endorphin and naloxone bind to Jurkat cell membrane preparations and can mutually displace each other from membrane binding sites. Tetraethylammonium ion, a potassium channel blocker, competitively displaces beta-endorphin and naloxone from membrane binding sites. Mitogen stimulated calcium ion flux is inhibited by tetraethyl ammonium and this inhibition is relieved by naloxone. With data derived from whole cell calcium ion flux studies, we accurately calculated the competitive displacement of beta-endorphin and naloxone from membrane preparations by tetraethylammonium thus showing that the action of these agents on potassium channels does not require second messengers. Using the resuspension induced ion flux technique, we find that beta-endorphin competes against naloxone for binding to Jurkat cells and naloxone competes against charybdotoxin, a potassium channel inhibitor, which like tetraethylammonium, is known to bind to the outer vestibule of the channel. Patch clamp electrophysiological studies show that beta-endorphin and naloxone exert complex actions on potassium channels in the presence or absence of mitogens. We conclude that one molecule of beta-endorphin or naloxone, but not both at the same time, bind to an area near the charybdotoxin/tetraethylammonium binding locus of Jurkat potassium channels.

Ultrasonification of biological materials: I. Apparatus

Abstract A self-aligning apparatus for use with a sonifier is described that permits reproducible ultrasonic treatment of solutions. The apparatus also reduces exposure of personnel to high-intensity sound.