Assessment of Fc (IgG) receptor function in adherent murine peritoneal macrophages using soluble model immune complexes

Fc (IgG) receptor function on thioglycolate-elicited adherent peritoneal macrophages from normal mice (C3H/HeN) and mice with abnormal activation of macrophages (C3H/HeJ) was studied. For this, soluble model immune complexes composed of five to six mouse anti-dinitrophenyl (DNP) IgG antibodies (heavy oligomers) were incubated with adherent macrophages cultured for either 2 or 48 hr. Cells from both strains bound similar amounts of oligomers at both 2 and 48 hr of culture (about 10⁶ IgG protomers/cell). Uptake of oligomers measured at 37 °C was also similar at 2 and 48 hr of culture. Endocytosis of oligomers occurred rapidly with about 50% of surface-bound complexes being internalized within 30 min, but there was no evidence for catabolism of the endocytosed material. There was a 50% decrease in the ability of macrophages to bind oligomers following a prior exposure to soluble complexes. Return to maximal binding after the preincubation with soluble complexes was incomplete for cells of both strains at both 2 and 48 hr of culture even after 2 hr at 37 °C.     

Regulation of DNA synthesis in mouse macrophages. II. Studies on mechanisms of action of the macrophage growth factor

When mouse macrophages are incubated with medium conditioned by mouse fibroblasts, they are induced to synthesize DNA and divide. This phenomenon is triggered by a macrophage growing factor (MGF) released by the fibroblasts. The presence of a serum cofactor is essential to the activity of the MGF; this cofactor can be removed by dialysis and seems unrelated to the “growth-promoting substances” normally present in serum. The kinetics of DNA synthesis in macrophages stimulated by conditioned medium (CM) is characterized by a lag phase of 24–48 h and a peak synthesis at 4–5 days, followed by a rapid decrease. This decrease is caused by depletion of the MGF from the CM by the growing macrophages.     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Differential scanning calorimetry on mixtures of lecithin,lysolecithin and cholesterol

The effect of increasing concentrations of lysolecithin (1-palmitoyl-sn-glycerol-3-phosphorylcholine) on the gel → liquid crystal thermal transition of lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine) in the aqueous phase was studied by differential scanning calorimetry (DSC). Lysolecithin showed an endothermic transition at 3.4°C whereas the transition of the lecithin occurred at 42°C. No phase separation could be observed calorimetrically at lysolecithin concentrations up to 60 mol%. Freeze etch electron microscopy showed that mixtures containing as much as 50 mol% lysolecithin exist in a lamellar phase. The lysolecithin was found to cause an initial slight increase in the enthalpy of transition followed by a gradual decrease. The enthalpy increased again at very high lysolecithin concentrations. The lysolecithin also caused a non-linear decrease in the temperature at which the lecithin transition took place.Cholesterol was found to decrease the enthalpy of transition of the lysolecithin, eliminating it at a concentration of 50 mol%. Cholesterol caused an increase in the temperature at which the lysolecithin transition took place.     

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.     

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon^® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.     

Rotational correlation times and partition coefficients of a spin label solute in lecithin vesicles

Di-tert-butylnitroxide dissolved in an aqueous suspension of egg yolk lecithin vesicles is distributed between the two phases. Partition coefficients of the nitroxide between the lipid and the water, calculated from the nitroxide electron paramagnetic resonance (EPR) spectra, decrease with decreasing temperature until approximately the freezing point of the solvent. Below this temperature the nitroxide is detected only in the lecithin. The rotational correlation times of the spin label present in the lecithin were calculated for the temperature range from +45 to ?60 °C. At low temperatures, the EPR spectra are characteristic of a superposition of two spectra resulting from the nitroxide dissolved in the lipid in two environments with different rotational correlation times.     

The ternary phase diagram of lecithin, cholesteryl linolenate and water: phase behavior and structure

The ternary phase diagram of cholesteryl linolenate-egg lecithin-water has been determined by polarizing light microscopy, calorimetry and X-ray diffraction at 23 °C. Hydrated lecithin forms a lamellar liquid-crystalline structure into which small amounts of cholesteryl linolenate are incorporated. The maximum incorporation of cholesterol ester into this lamellar structure varies with the degree of hydration. Increasing the water concentration from 10 to 15% (w/w) increased the limiting molar ratio of cholesteryl linolenate to lecithin in the lamellar phase from 1:50 to 1:22. At intermediate concentrations (15 to 30% water) the cholesteryl linolenate:lecithin ratio remains constant at 1:22. When water is increased to 42.5%, the maximum water content in the lamellar phase, the molar ratio decreased to 1:32. At low water concentrations the cholesterol ester appears to be entirely in the apolar region of the lecithin bilayer, while at higher water concentrations the ester groups of cholesteryl linolenate may be located at the lipid-water interface. At high water concentrations the ester appears to disorder the alkyl chains of the lecithin, giving rise to a thinner lipid layer and an increased surface area per lipid molecule when compared to the lecithin-water system in the absence of cholesteryl linolenate.The lamellar phase is the only phase (except at water concentrations less than 5%) in which all three components mutually interact. All mixtures of the three components having compositions outside the one-phase (lamellar) zone produce additional phases of cholesteryl linolenate or water, or both. Between 23 °C and 60 °C only minor changes in the phase diagram are observed.     

Solvent-free enzymatic hydrolysis of non-polar lipids in crude sunflower lecithin using phospholipase A1 (Lecitase® Ultra)

In this contribution, a new chromatographic method was applied to study the hydrolysis of non-polar lipids, i.e. triacylglicerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG), when crude sunflower lecithin is treated with Lecitase^® Ultra, an enzymatic preparation with phospholipase A₁ (PLA₁) activity. Results not only proved the enzyme lipase activity toward non-polar lipids in selected reaction conditions (aqueous system, T?=?50?°C, pH?=?5) but also suggested the occurrence of acyl-migration phenomenon observed by other authors in similar systems. Results showed that 1?h of reaction was enough to decrease the content of TAG in 54%, while DAG and MAG concentration increased from 0.4 to 3.5 and from 1.9 to 6.5?g/100?g of crude lecithin, respectively. Along the reaction, different contents of glycerides could be achieved, obtaining products with different composition which, in combination with the presence of phospholipids (PL) and/or lysophospholipids (LPL), could present specific emulsifying/stabilizing properties with a wide range of applications in food and pharmaceutical industry.     

Effect of incubation temperature after devitrification on quality parameters in human sperm cells

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.     

Factors influencing the pathogenicity and development of Mattesia trogodermae infecting Trogoderma glabrum larvae

Factors that regulate development of Mattesia trogodermae in Trogoderma glabrum were defined, and their quantitative effects were determined. The rate of and the extent to which spore formation proceeds is strictly governed by temperature. More spores are produced at 30° than at 25°C and very low numbers of spores are formed when the incubation temperature is 35°C. When insects are incubated at 35°C for 1–10 days and transferred to 30°C for the remainder of the 30-day experiment, spore production capacity gradually declines with increasing time at 35°C. Two hypotheses are proposed for this phenomenon. Larval size also regulates the extent of spore production, larger larvae having greater potential for spore development. This is not influenced by dosage. Spore production in pupae and adults was always retarded.Dosage and environmental conditions which influence the virulence of M. trogodermae were investigated. These studies show that rates of mortality are higher at higher temperatures. Low doses of spores result in longer LT50's than do high doses at 25° and 30°C. No differences in rates of mortality were found between different doses at 35°C.     

The antiteratogenic effects of hypo- and hyperthermia in trypan blue-treated chick embryos

Fertile White Leghorn chicken eggs (N = 174) were incubated under optimum conditions until the embryos had reached Hamburger-Hamilton stage 12 (about 48 hr). At that time, 20 μl of 1% trypan blue solution, dissolved in 0.85% NaCl (wt/vol) was injected through the yolk sac into the liquid yolk found just under the embryo. After injection, the eggs were separated into groups and returned to the incubator under control conditions (38°C), or at temperatures lower (35°C) or higher (41°C) than optimum.After an additional 24 hr of incubation, the embryos incubated at 35°C (N = 53) exhibited significantly fewer caudal hematomas (P < 0.02) than did embryos incubated at 38°C (N = 51). Similarly, embryos incubated at 41°C (N = 40) also exhibited significantly fewer caudal hematomas (P < 0.05) than did their corresponding (38°C) controls (N = 30). There was no significant difference between the 35°C group and their controls, or the 38°C group and their controls, in embryonic dry weight, dry weight of the area vasculosa, or crown-rump length. The only other significant difference detected between groups was a very slight but significant (P < 0.0005) decrease in Hamburger-Hamilton stage (0.4 stage unit) between embryos incubated at 35°C and the corresponding controls.Since incubation temperatures either above or below optimum result in a marked reduction in the teratogenic response to trypan blue treatment, we conclude that there exists a temperature optimum for the development of caudal hematomas.     

Nuclear magnetic resonance studies of amphiphile hydration: Effects of the gel-to-liquid crystalline phase transition on the hydration of dioleoyl lecithin

The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between ?15 and ?30 °C, using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/mol of phospholipid at ?25 °C (our “standard” temperature) for DOL, and only 6 mol of water/mol of phospholipid for DML. The gel-to-liquid crystalline phase transition in DOL centered at ca. ?19 °C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.     

Heart rate during development in the turtle embryo: effect of temperature

 Growth and development can occur over a wide range of physical conditions in reptiles. Cardiovascular function must be critical to this ability. However, information on cardiovascular function in developing reptiles is lacking. Previous work indicated that in reptiles the effects of temperature on growth and metabolism are largely restricted to early development. This study examined whether the previously observed effects of temperature and different perinatal patterns of metabolism observed in amniotic vertebrates are correlated with cardiovascular function. Embryonic and hatchling carcass mass, heart mass and heart rate (HR) were compared for snapping turtle eggs (Chelydra serpentina) incubated at 24 ° and 29 °C. Incubation time was shorter at 29 °C (56.2 days) than at 24 °C (71.1 days). Carcass and heart growth showed a sigmoidal pattern at both temperatures. However, cardiac growth showed a relative decrease as incubation proceeded. Incubation temperature significantly affected the HR pattern during development. The HR of embryos incubated at 24 °C was constant for most of incubation (51.8±4.8 min^-1). A small decrease was observed just prior to and a large decrease immediately following hatching (posthatch, 22.3±4.1 min^-1). At 29 °C embryonic HR was greater than at 24 °C early in development (72.3±3 min^-1). The HR steadily decreased to values equivalent to those at 24 °C. The HRs of 24 °C and 29 °C hatchlings were not different. Cardiac output (estimated as the product of heart mass and HR) increased rapidly during early development and then slowed dramatically at both temperatures. These data are consistent with the suggestion that temperature exerts its effects primarily early in development. Furthermore, the changes in cardiovascular function are correlated with metabolic changes in hatching vertebrates. Accepted:12 June 1996     

Temperature sensitivity of protein synthesis initiation. Inactivation of a ribosomal factor by an inhibitor formed at elevated temperatures.

When a reticulocyte lysate, supplemented with hemin, was warmed at 42 °C, its protein-synthesizing activity was greatly decreased. This was accompanied by the reduced formation of the 40 S·Met-tRNA_f initiation complex. This complex preformed at 34 °C, however, was stable and combined with added globin mRNA and the 60 S ribosomal subunit to form the 80 S complex at the elevated temperature. When the ribosome-free supernatant fraction of lysates was warmed at 42 °C with hemin and then added to the fresh lysate system, it inhibited protein synthesis by decreasing the formation of the 40 S complex. This decrease in protein synthesis by warmed lysates or warmed supernatant could be overcome by high concentrations of GTP and cyclic AMP. This effect of GTP and cyclic AMP was antagonized by ATP. The results indicate that the inactivation of protein synthesis by the lysate warmed at 42 °C is due to the formation of an inhibitor in the supernatant. The ribosomal KCl extract prepared from the lysate that had been warmed at 34 °C and then incubated at this temperature for protein synthesis supported protein synthesis by the KCl-washed ribosome at both 34 and 42 °C. On the contrary, the extract from lysates that had been warmed at 42 °C and then incubated at 34 °C could not support protein synthesis at 42 °C, although it was almost equally as promotive as the control extract in supporting protein synthesis at 34 °C. The results indicate that the factor which can protect protein synthesis against inactivation at 42 °C is itself inactivated in lysates warmed at 42 °C. However, the activity of this extract to support formation of the ternary complex with Met-tRNA_f and GTP was not reduced. Native 40 S ribosomal subunits isolated from lysates that had been warmed at 42 °C and then incubated for protein synthesis indicated that the quantity of subunits of density 1.40 g/cm³ in a CsCl density gradient were decreased while those of density 1.49 g/cm³ were increased. The factor-promoted binding of Met-tRNA_f to the 40 S subunit of lower density from the warmed and unwarmed lysates was equal, suggesting that the ribosomal subunit was not inactivated. These results were discussed in terms of the action of the inhibitor formed in the supernatant at 42 °C, which may inactivate a ribosomal factor essential for protein synthesis initiation.     

Reversal of embryonic diapause in the Australian plague locust, Chortoicetes terminifera (Walker), by temperatures above the development threshold

Diapause was induced in embryos of Chortoicetes terminifera (Walker) by transferring adults from an L:D 15:9 regime to an L:D 12.5:11.5 regime. When incubated at 20°C all eggs in all pods entered and remained in diapause but when incubated at 26, 32 and 38°C a proportion of eggs in some pods did not. Pods incubated at 32°C for up to 6 days, when diapause intervenes and then transferred to 20°C gave the same result as pods incubated at 20°C throughout development. All eggs entered and remained in diapause. If the period at 32°C was extended to 8 days, the proportion remaining in diapause was not significantly different from that found when pods were incubated at 32°C throughout development. In eggs which broke diapause at 32°C there was a pause or slowing down of development for about 2 or 3 days around the stage at which diapause intervenes.     

Turnover of murein in cellular and filamentous populations ofBacillus megaterium

Bacillus megaterium grows in the form of filaments at temperatures above 45°C. The rate of turnover of the cell wall begins to decrease gradually under these conditions. At the same time sensitivity of the filamentous forms to lysozyme decreases. Filaments outgrown at 48°C retain the decreased rate of turnover of the cell wall for a certain time after transfer to 30°C, in spite of the fact that septa are formed and filaments are converted to cells. However, a population incubated longer than 2 h at 48°C often ceases to grow and the growth is not restored even after transfer to 30°C. Three clones of the asporogenic strainBacillus megaterium KM differing somewhat in their ability to form filaments at 35°C differ mutually also in the rate of turnover of the cell wall. However, the decreased rate of the turnover cannot be unambiguously correlated with the increased tendency to form filaments.     

Studies on fluorescence polarization of 1-acyl-2-cis- or trans-parinaroyl sn-3-glycerophosphorylcholines in model systems and microsomal membranes

Fluorescent lecithin probes containing cis- or trans-parinaric acid (PnA) at the 2-position cis-parinaroylphosphatidylcholine (cis-PnPC) and trans-parinaroyl phosphatidylcholine (trans-PnPC)) showed similar behavior to that of the free cis- or trans-parinaric acids (cis-PnA or trans-PnA) in bilayer vesicles of synthetic saturated lecithins. Transition temperatures detected by cis-PnPc were about 1°C lower than those observed with trans-PnPc. In mixed lecithin vesicles, the trans-PnPc probe monitored a higher temperature melting component than did the cis-probe. Both probes were readily incorporated into microsomal membranes and into sonicated vesicles prepared from the microsomal phospholipids. With either cis- or trans-PnPc no change in polarization ratio was observed for microsomal membranes between 40°C and 0°C but this ratio increased with decreasing temperature between 0°C and ?5°C. However, vesicles of extracted phospholipids showed a continuous increase in polarization ratio with decreasing temperature between 20°C and ?15°C with trans-PnPc and bewteen 5°C and ?15°C with cis-PnPc. These results suggest that the two lecithin probes monitor different environments in the membranes and phospholipid vesicles prepared from them.     

Effects of incubation temperature on the organic amendment‐mediated control of Fusarium wilt of tomato

Organic soil amendments play important roles in the reduction of plant diseases caused by soil‐borne plant pathogens. This study examined the combined effects of concentrations of organic amendments, temperature and period of incubation in soil on the management of Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (Fol). In an experiment with substrate mixture, Fol reduction was higher when the soils were incubated at 35°C than at 30°C. Disease severity was proportionally reduced as the volume of amendment added increased. Furthermore, disease was significantly lower in substrates incubated for 30 days at both temperatures, as compared to substrates incubated for only 15 days. The most effective control was achieved with pelletised poultry manure (PPM). In experiments with natural sandy soil, the effects of amendments on Fol populations, measured by real‐time quantitative PCR with TaqMan probes, were significant. The highest decreases in Fol DNA resulted when the soil was amended with 2% PPM and incubated at 35°C. The reductions in DNA concentrations was most likely related to the accumulations of high concentrations of NH₃ (27.3 mM) in soils treated with 2% PPM and incubated at room temperature (RT; 23 ± 2°C), or at 35°C. Severity of plants grown in soils incubated at RT decreased by over 40%, and more than 73% when incubated at 35°C, regardless of the rate of PPM. The results indicate that the management with PPM, when combined with heating or solarisation, is an effective control measure against Fusarium wilt of tomato.     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.