Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

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.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

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.     

Differential scanning calorimetry and enzymic activity of rat liver microsomes in the presence and absence of Δ1-tetrahydrocannabinol

The thermal transitions of rat liver microsomes and isolated lipids were investigated by using differential scanning calorimetry. Endothermic transitions at ≈?5°C and between ≈18° and 40°C were detected in the membranes and at ≈?10°C and between ≈10° and 20°C in the extracted lipids.Interaction with $\text{Δ}{}^1\text{-}\text{tetrahydrocannabinol}$ of microsomal membranes and of extracted lipids influences the thermotrophic behaviour as revealed by differential scanning calorimetry and eliminates the break in the Arrhenius plot of the enzymic activity of $\text{O-}\text{demethylase}$.     

Pharmacological properties of imidazole. 2. Action on the normal body temperature regulation in rats

Rats injected intraperitoneally with imidazole (100 to 400 mg/kg) showed a progressive dose-dependent decrease in rectal temperature. A decrease of 6$\text{°}$C occurred when the imidazole-treated animals were placed in a 4$\text{°}$C environment. In a 23$\text{°}$C environment, the rectal temperature declined about 2$\text{°}$C. The results show that imidazole induces a decline in body temperature in non-febrile rats, possibly due to an action on hipothalamic calcium levels.     

Isolation and characterization of minor glycosaminoglycans in the rabbit vitreous body

Protein synthesis in the microvascular system of the rabbit brain was inhibited following the elevation of body temperature by 2?2.5°C. $\text{In}$,$\text{vivo}$ labeling studies indicated that hyperthermia also induced the synthesis of a 74K protein in cerebral microvessels which is similar in molecular weight to one of the major heat shock proteins previously reported in tissue culture systems following elevation of ambient temperature. The present results suggest that a physiologically relevant increase in body temperature, similar to that attained during fever reactions, can induce the synthesis of a heat shock protein in the cerebral microvascular system of the intact mammal.     

Properties and structure of a gene 24-controlled T4 giant phage

Giant T4 bacteriophage were found by Doermann et al. (1973a) with point mutants in gene 23 and by Cummings et al. (1973) after l-canavanine induction followed by an arginine chase. We now find T4 giant phage with 14 out of 15 tested temperature-sensitive mutants in gene 24 grown at intermediate temperatures between 33 °C and 37 °C.For one of these mutants, T4,24(tsB86), we found that (a) the optimum temperature for giant phage production is 34.8 °C, (b) the head-length distribution peaks sharply between 10 and 12 normal T4 phage head lengths, (c) about 75% of our giant phage have two tails, (d) the buoyant density in CsCl is greater than that of normal phage, (e) they are infectious and show an increased u.v. resistance, (f) their sodium dodecyl sulphate gel electrophoresis pattern is qualitatively similar to that of normal T4 phage, although the relative intensities of some of the bands are different, showing for example, a decreased $\text{P24}{}^1\text{P23}{}^1$² ratio, (g) optical diffraction and filtering of the flattened cylindrical part of the giant heads show a p6 surface net with a lattice constant of approximately 130 Å, a unique $\text{u}\text{v}$ ratio of $\text{15}\text{5}$ and a capsomer morphology of the type 1 + 6 + 6.Mixed infections with T4 wild type and T4.24(amN65) also yield giant phage. These are produced in highest amounts with a multiplicity of infection ratio of 5:5; no giants are observed at ratios of 1:9 or 9:1, suggesting that their formation may be caused by a dosage effect of P24.     

Activity of endonuclease S1 in denaturing solvents: dimethysulfoxide, dimethylformamide, formamide and formaldehyde.

The endonuclease S₁ from $\text{Aspergillus oryzae}$ is shown to be active in aqueous solutions of greater than 60 percent formamide, 50 percent dimethylsulfoxide, 30 percent dimethylformamide and in 2 percent formaldehyde. The melting profile of T7 DNA is determined by S₁ digestion in 50 percent formamide and is found to agree with the optical melting profile. The single-strand specificity of S₁ is retained in 50 percent formamide at 37°C, 3°C below the DNA melting temperature. S₁ is shown to be active at temperatures greater than 5°C above the DNA melting temperature in this solvent.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

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.     

Administration of heparin causes in vitro release of non-esterified fatty acids in human plasma

The objective of this study was to investigate the extent to which $\text{in vitro}$ hydrolysis of endogenous triglycerides contributes to the elevated concentrations of non-esterified fatty acids (NEFAs) which have been reported after heparin administration. Heparin is known to induce the release of lipases which hydrolyze endogenous substrate both $\text{in vivo}$ and $\text{in vitro}$. Four patients undergoing diagnostic cardiac catheterization, who routinely receive heparin, were studied. Blood samples were obtained before and at 5 and 30 minutes after an intravenous bolus of heparin (46 U/kg) was administered. Determinations of NEFAs in plasma were carried out immediately and again various times after the samples had incubated at 24°C and at 0°C. In addition, an aliquot of each sample was frozen quickly, stored for 5–7 days, thawed, and incubated at 24°C for 180 minutes. As expected, there were no significant increases after incubation in the concentrations of NEFAs in the samples obtained before heparin administration. In contrast, in the samples obtained after heparin administration, incubation at 24°C produced significant increases in the concentrations of NEFAs. For example, in the plasma samples obtained 5 minutes after administration of heparin, concentrations of NEFAs increased 50, 160 and 300% after 5, 60, and 180 minutes of incubation compared to pre-heparin concentrations. When assayed immediately, the concentrations of NEFAs increased only 15% over pre-heparin concentrations. Incubating the samples at 0°C slowed lipase activity. Freezing the samples stopped the lipase activity; however, when the thawed samples were incubated at 24°C, concentrations of NEFAs continued to rise. This study suggests that much of the reported increases in the $\text{in vivo}$ concentrations of NEFAs after administration of heparin may be due to $\text{in vitro}$ formation from continued lipase activity on endogenous substrate. Moreover, studies relating increases in the concentrations of NEFAs after administration of heparin to changes in drug binding to plasma proteins should be re-examined for possible $\text{in vitro}$ artifacts.     

Kinetics of repair of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver $\text{in}\text{,}\text{vivo}$ occurred at rates comparable to those seen $\text{in}\text{,}\text{vitro}$ with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal $\text{in}\text{,}\text{vivo}$ and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.     

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.     

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

A single peak of DNA polymerase activity from extracts of $\text{T.}\text{brucei}$, obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg²⁺ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)_12–18. Due to the potential significance of polyamines in the metabolism of $\text{T.}\text{brucei}$, the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.     

The stability of L-phenylalanine mustard in bile

L-phenylalanine mustard (L-PAM) was incubated at 37° C in bile of bovine, canine and human origin. Recovery rate constants of L-PAM from bile were 0.1/hr for canine bile (0–3 hours); 0.18/hr for bovine bile; 0.45/hr for human bile. No significant hydrolysis of L-PAM in canine bile was noted for the period of 3 to 6 hours at 37° C. The incubation of L-PAM in sodium taurocholate solution (1000 molar excess) gave a recovery rate constant 0.15/hr at 37° C. However, the incubation of L-PAM in bilirubin solution (2.5 mg/ml H₂O) gave a recovery rate constant of 0.52/hr at 37° C. The high concentration of the parent compound L-PAM seen $\text{in vivo}$ in canine bile after i.v. administration may be related to its low $\text{in vitro}$ degradation rate in canine bile.     

Lethal heat induces single strand breaks in the DNA of bacterial spores

Lethal heating induces DNA single strand breakage in bacterial endospores as detected by the alkaline sucrose gradient centrifugation technique. Heating of spores of $\text{Bacillus}\text{subtilis}$ 168 at 90°C for 10, 30, and 60 min induced 6, 15, and 15 single strand breaks, respectively and inactivated 6%, 98.2%, and 99.974% of the spores. This is the first report to our knowledge identifying specifically single strand DNA breakage with lethal heat injury of bacterial spores.     

Melting behaviour of a triple helical polysaccharide schizophyllan in aqueous solution

Two sonicated samples of schizophyllan in aqueous solution at temperatures from 20 to 160°C were investigated by viscometry. The temperature dependence of the viscosity coefficient η showed that schizophyllan in water undergoes an irreversible thermal transition at about 135°C. The values of $\text{(ln η}{}_{\mathrm{r}}\text{)}\text{c}$ (ηr is the relative viscosity and c is the polymer concentration (w/v)) at 25°C determined after preheating aqueous schizophyllan indicated that the major conformations of schizophyllan in water at 120 and 150°C are triple helix and single random coil, respectively. Thus, it was concluded that the change in η at about 135°C with an increase in temperature is due to the melting of triple helices to single chains. Schizophyllan denatured to single chains at about 150°C did not restore the intact triple helix, but formed aggregates, when the solution was cooled to 25°C. It was also found that the aggregates form a gel when c is higher than a certain value.     

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.