Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2

Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.     

Molecular dynamics simulations of "loop closing" in the enzyme triose phosphate isomerase

We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 A during the simulation, some residues in the active site loop move more than 8 A during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to "close off" the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.     

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.     

Evidence for lactate utilization for fetal lung glycogen synthesis

Fetal rabbit lungs from 23 day gestation animals were used to investigate the potential role of lactate as a substrate for fetal lung glycogen synthesis. Fetal lactate dehydrogenase activity was approximately twice that found in the adult lung, while the activity of phosphoenolpyruvate carboxykinase was elevated fourfold over the adult value. Pyruvate carboxylase activities were similar in both fetal and adult lungs. Studies employing fetal lung explants in organ culture indicated that the presence of both glucose and lactate may be necessary for glycogen accumulation in the developing fetal lung. These data support the hypothesis that lactate is an important precursor for fetal lung glycogen.     

1,2-Dimethylhydrazine-induced alterations in colonic plasma membrane fluidity: restriction to the luminal region

Recently, work in this laboratory has shown that changes in the 'dynamic' component of fluidity, lipid composition and phospholipid methylation activity of distal colonic brush-border membranes could be detected after administration of 1,2-dimethylhydrazine to rats of the Sherman strain for 5-15 weeks, i.e., before the development of colon cancer. The present experiments were therefore conducted to: determine whether similar 'premalignant' biochemical changes could be detected in basolateral membranes of Sherman rats treated with this agent; and clarify the relationship of these membrane changes to the malignant transformation process by examining the effect of 1,2-dimethylhydrazine on these biochemical parameters in colonic antipodal plasma membranes of rats of the Lobund-Wistar strain. This particular strain of rats has previously been shown to be total resistant to the induction of tumors by 1,2-dimethylhydrazine. The results of the present experiments demonstrate that similar biochemical alterations could not be detected in the colonic plasma membranes prepared from either strain of rat treated with 1,2-dimethylhydrazine. These data support the contention that the prior biochemical membrane alterations noted in brush-border membranes of 1,2-dimethylhydrazine-treated animals are, in fact, related to the malignant transformation process and, furthermore, are confined to the luminal surface of distal colonic epithelial cells.     

Beta-glucosidase from Trichoderma reesei. Substrate-binding region and mode of action on [1-3H]cello-oligosaccharides

To determine the mode of action of the beta-glucosidase from Trichoderma reesei a method was developed for synthesizing [1-3H]cello-oligosaccharides with specific radioactivities of approximately 3000 Ci/mol. The beta-glucosidase removed glucosyl residues from the non-reducing end of the [1-3H]cello-oligosaccharides in a multiple attack mode with little tendency to attack the substrates repetitively. Values of Km were lower for longer cello-oligosaccharides, whereas values of V remained essentially constant. A subsite map, constructed using values of V/Km for the cello-oligosaccharides, showed that the substrate-binding region comprises primarily three subsites.     

Convective and radiative components of wind chill in sheep: Estimation from meteorological records

Wind chill is defined as the excess of sensible heat loss over what would occur at zero wind speed with other conditions unchanged. Wind chill can be broken down into a part that is determined by air temperature and a radiative part that comprises wind-dependent effects on additional long-wave radiative exchange and on solar radiation (by reducing solar warming). Radiative exchange and gain from solar radiation are affected by changes that are produced by wind in both surface and fleece insulations. Coefficients are derived for (a) converting the components of sensible heat exchange (air-temperature-dependent including both convective and associated long-wave radiative, additional long-wave radiative and solar) into the components of the total heat loss that are associated with wind and (b) for calculating equivalent air temperature changes. The coefficients contain terms only in wind speed, wetting of the fleece and fleece depth; these determine the external insulation.Calculation from standard meteorological records, using Plymouth and Aberdeen in 1973 as examples, indicate that in April–September 1973 at Plymouth reduction in effective solar warming constituted 28% of the 24-h total wind chill, and 7% in the other months of the year combined; at Aberdeen the corresponding percentages were 25% and 6%. Mean hour-of-day estimates for the months of April and October showed that at midday reduction in solar warming due to wind rose to the order of half the air-temperature-dependent component of wind chill, with a much smaller effect in January. For about six hours at midday in July reduction in solar warming due to wind was similar in magnitude to the air-temperature-dependent component.It is concluded that realistic estimates of wind chill cannot be obtained unless the effect of solar radiation is taken into account. Failure to include solar radiation results not only in omitting solar warming but also in omitting the effects of wind in reducing that warming.The exchange of sensible (non-evaporative) heat loss between a homeothermic animal and its environment can be divided into two parts: one part is due to the temperature difference between the animal and the surrounding air, and the other part is due to additional long-wave radiative exchange between animal and environment and to solar radiation. Both parts of the heat exchange are determined in magnitude by the animal's thermal insulation, which is itself affected by windspeed and wetting. Wind diminishes as animal's external insulation, so increasing heat loss under all conditions when the air temperature is lower than the animal's surface temperature: this effect is termed wind chill. Wind chill has previously been investigated more commonly in relation to man (Burton an Edholm, 1955; Smithson and Baldwin, 1978; Mumford, 1979; Baldwin and Smithson, 1979). This paper is concerned with the separate contributions to wind chill calculated for sheep that can be associated with convective and radiative heat exchanges.