The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

The temperature dependence of chain disorder in potassium palmitate-water. A deuterium NMR study

Quadrupolar echo deuterium magnetic resonance spectroscopy is used to study the hydrocarbon chain disorder in a 70% potassium palmitate-30% water mixture. The C-D bond order parameters are measured as a function of temperature and position along the chain. In the liquid crystalline, L_α, phase the C-D bond order parameters of the methylene segments near the polar head group are found to increase with increasing temperature to a maximum at about 100°C and then decrease on further increasing the temperature. The order parameters for the remaining methylenes and the methyl group decrease monotonically with increasing temperature. The C–D bond order parameter for the α position undergoes a discontinuous change at $\text{～55°}\text{C}$ and, in fact, for a 10°C range two resolvable splittings are observed for the α posittion. The low temperature, coagel, phase gives a well resolved spectrum where all methylenes are equivalent yielding a value of 168 kHz for e²qQ/h, the quadrupole coupling constant.     

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements.

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus $\text{1}\text{T}$ was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range.Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) $\text{J. Mol. Biol. 85}$, 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.     

Behavioral fever in anuran amphibian larvae.

Following intraperitoneal injection with killed gram-negative bacteria, $\text{Aeromonas}$$\text{hydrophila}$, tadpoles of $\text{Rana}$$\text{catesbeiana}$ and of $\text{R}$. $\text{pipiens}$ showed significant mean increases in preferred temperature of 2.6°C and 2.7°C, respectively, in an electronic thermoregulatory shuttlebox device. This “behavioral fever” is similar to elevations in preferred temperature previously demonstrated for fishes, reptiles and mammals, although both normal and febrile thermal preferenda vary among vertebrates.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Detection of a phase transition in red cell membranes using positronium as a probe

Positron lifetimes in human red cell ghost membranes have been measured as a function of temperature from 3°C to 25°C. A marked sudden change in the $\text{ortho-}\text{positronium}$ annihilation rate was found at 16–18°C during the heating cycle and at 18–20°C in the cooling cycle. Such sudden change of microenvironment in the membranes sensed by $\text{ortho-}\text{positronium}$ is attributed to the sudden change of water diffusion rate through the membranes which is a consequence of the sudden change in free volume, or fluidities in the lipid layers.     

Cellular pH and water permeability control in frog urinary bladder a possible action on the water pathway

Mucosal acidification (from pH 8.1 to 6.0) reversibly inhibited the hydroosmotic responses to oxytocin, cyclic AMP and 8-bromo-cyclic AMP in frog urinary bladder. These inhibitory effects were only observed in the presence of a permeant buffer in the apical medium and could also be elicited by CO₂ bubbling, even when the mucosal pH was clamped at 8.1. Acid pH reduced the oxytocin-induced net water flux faster than norepinephrine or oxytocin removal and the difference was especially important at low temperature. The time course of recovery from acid pH inhibition was, at 20°C, similar to that of the hormonal action, but when the medium temperature was reduced to 6–7°C, the recovery from acid pH inhibition paradoxically became faster while the oxytocin action was markedly slowed down ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of changes in net water fluxes (expressed in min): oxytocin addition at 20°C, $\text{6.2 ± 0.9}$; at 6°C, $\text{24 ± 3}$; oxytocin removal at 20°C, $\text{4.7 ± 0.8}$; at 6°C, $\text{22 ± 3}$; pH inhibition at 20°C, $\text{2.6 ± 0.2}$; at 6°C $\text{2.5 ± 0.2}$; recovery from pH 6 at 20°C, $\text{6.5 ± 0.9}$; at 6°C, $\text{2.7 ± 0.3}$). These results can be explained by accepting two main loci sensitive to medium acidification: (1) the cyclase system and (2) an intracellular, temperature-independent, post-cyclic AMP site. The fact that the intramembranous particle aggregates associated with the oxytocin-induced water permeability increase did not disappear after the flow inhibition by acid pH at low temperature suggests that the second effect could be located at the water channel itself.     

Seasonal and temperature-related changes in mitochondrial membranes associated with torpor in the mammalian hibernator Spermophilus richardsonii

Seasonal variations in the thermal response of liver mitochondrial membranes from Richardson's ground squirrels (Spermophilus richardsonii) were determined by measuring succinate-cytochrome $\text{c}$ reductase activity and spin label motion over a temperature range of 2 °C to 35 °C. For seven summer animals from the field the Arrhenius-type plots for enzyme activity and spin label motion were biphasic indicating a transition in structure and function at $\text{22 + 2.3°C}\text{and}\text{23 ± 1.9°C}$, respectively; typical of homeothermic mammals. For 12 winter animals maintained at 19°C, the transition in structure and function was lowered to $\text{12 ± 1.1°C}$ and $\text{13 ± 1.4°C}$, respectively. The transition for 5 of 11 winter animals which were kept at 4°C and maintained normal activity and body temperature was similar to animals maintained at 19°C, while for the other six the transition was further lowered to less than 4°C. The transition for seven winter animals which were in deep hibernation was less than 4°C. The results for liver mitochondria show that lowering of the transition in membrane structure and function occurs as a two-stage process of about 10 deg. C for each stage and that the lowering is a requisite for hibernation rather than a response to the low-body temperatures experienced during hibernation.     

Effect of chronic heat exposure on in-vitro drug metabolism in the rat

The $\text{in-vitro}$ rate of drug metabolizing enzymes was investigated in chronically heat exposed male rats. Exposure to 35±2°C for 30 days resulted in significant reduction in the rates of both hepatic N-demethylation of parachloro-N-methylaniline and aniline hydroxylation. Substantial elevation of rectal temperature and marked reduction in body and liver weights was observed in the same rats. The significance of these results is discussed.     

Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles

The temperature dependence of the binding of PhNapNH₂ ($\text{N-}\text{phenyl-1-naphthylamine}$) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 °C, some 30 °C above the broad phase transition region of the phospholipid. In the temperature range above 20 °C, $\text{ΔH}{}^0\text{= ?6100}\text{cal·mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 9.7}\text{e. u.}$; in the temperature range below 20 °C, $\text{ΔH}{}^0\text{= 0}\text{cal · mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 30.4}\text{e. u.}$ These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 °C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 °C, though significantly greater than that above 20 °C, is still significantly less than that in the crystalline state.     

Effect of heat on frog sciatic nerve determined by X-ray diffraction

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerves in Ringer's solution after heat treatment from 20 to 80°C. The X-ray patterns were obtained from the heat treated specimens after cooling to room temperature. The normal X-ray pattern of frog sciatic nerve in Ringer's solution with $\text{d=171}\text{A}\text{?}$ was maintained from 20 to 58°C. Above 58°C, a new high temperature pattern based on a repeat period of $\text{d?435}\text{A}\text{?}$ was recorded from the nerve in Ringer's solution. The physical state of nerve myelin after heat teratment at a temperature ?58°C has been identified as the anomalous swollen state. Anomalous swelling takes place in units of four membranes.     

The influence of cholesterol on head group mobility in phospholipid membranes

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0–70°C for various mixtures of $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency $\text{f}{}_2$ observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in $\text{f}{}_2$ are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, a decrease in $\text{f}{}_2$ at cholesterol contents up to 20–30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.     

Investigation of human erythrocyte ghost membranes with intramolecular excimer probes

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37°C to 570 cP at 5°C, as reported for di(1-pyrenyl)propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37°C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40°C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol ($\text{? = 13.3}$) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol ($\text{? = 17.5}$), whereas a polarity comparable to that of methanol ($\text{? = 32.7}$) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.     

Cytochrome b5/cytochrome b5 reductase interaction in microsomes: kinetics at subzero temperature.

The cytochrome $\text{b}{}_5\text{b}{}_5$ reductase system solubilized from microsomes exhibits monophasic reduction kinetics over the temperature range 15 ° to ?25 °C in aqueous/ethylene glycol co-solvent, whereas in intact microsomes, the process becomes increasingly heterogeneous below 0 °C, reflecting heterogeneities in membrane structure observable as distributions in reaction rates and activation energies.     

Temperature dependence of active K+ transport in cation dimorphic sheep erythrocytes

Arrhenius diagrams of K⁺ pump fluxes measured between 15°C and 41°C were discontinuous in high K⁺ but not in low K⁺ sheep red cells. Exposure of low K⁺ cells to anti-L caused a bimodal temperature response of K⁺ pump flux with a transition temperature, $\text{T}\text{c}$, similar to that found in high K⁺ cells but with comparatively higher activation energies above $\text{T}\text{c}$.     

Evidence for cooperative Mn-ATP binding with Bacillus sp. glutamine synthetase

The binding of Mn-ATP with $\text{B. subtilis}$ glutamine synthetase, observed kinetically at 37°, pH 7.0, is cooperative (Hill $\text{n}\text{= 2.3, S}{}_{\mathrm{0·5}}\text{= 0.36mM)}$, a phenomenon overlooked in earlier studies. The Arrhenius plot is biphasic with a break at 26°C. Similar behavior is observed with the thermophilic $\text{B. stearothermophilus}$ enzyme, but is absent with the enzymes from $\text{E. coli}$, plant, and mammaliam sources under optimal assay conditions. The temperature dependence of the intrinsic fluorescence of the protein is also non-linear, and the intersection point of 18° shifts to 30° upon binding of substrates. These results are interpreted as indicating that $\text{Bacillus sp}$. enzymes can assume multiple, functionally important conformational states related to Mn-ATP binding at 37°. They also emphasize further that critical differences in mechanism exist among glutamine synthetases from different sources.