Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Temperature Elevation Regulates Iron Protoporphyrin IX and Hemoglobin Binding by Porphyromonas gingivalis

Porphyromonas gingivalis, an obligate anerobe with a growth requirement for iron protoporphyrin IX (FePPIX), is exposed to increased temperatures in the inflamed periodontal pocket. In this study, P. gingivalis was grown in a chemostat at 37°C (control), 39°C, and 41°C, and examined for hemagglutinating (HA) activity, hemoglobin binding and degrading activity, and iron protoporphyrin IX binding. HA activity decreased in cells as the growth temperature increased. Binding of μ-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39°C-grown cells but decreased in 41°C-grown cells compared with controls. Cellular hemoglobin binding and degradation decreased with increased growth temperature. The decrease in cellular hemagglutination and hemoglobin degradation occurring with increased growth temperature would limit the potential overproduction of toxic monomeric haem molecules. The increased binding of μ-oxo bishaem and monomeric forms of FePPIX at 39°C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance. Received: 24 April 2000 / Accepted: 30 May 2000     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

Seasonal patterns in the physiology of the European brown bear (Ursus arctos arctos) in Finland

The physiological indicators such as body temperature, blood chemistry and hematology of seven European brown bears (Ursus arctos arctos) were used in the present study. They were kept in either the Zoological Garden of University of Oulu (65°N, 25°24'E) or the Ranua Zoological Garden approx. 150 km NE of Oulu. Transmitters with a temperature-dependent pulse rate were implanted subcutaneously or into the abdominal cavity under anesthesia. Our data indicate that the body temperature of the bear decreases during the winter sleep to 4–5°C below the normal level (37.0–37.5°C). The lowest values, 33.1–33.3°C, were measured several times in midwinter. Hematocrit, hemoglobin and erythrocyte counts seem to be higher, and the leucocyte count lower during the denning period than in the awake bear. Plasma N-wastes were lower during the winter sleep than before or after it. The analysed blood parameters showed that plasma catecholamines and thyroid hormones decreased in the fall.     

Mitochondrial function in Antarctic notothenioid fishes that differ in the expression of oxygen-binding proteins

State III respiration rates were measured in mitochondria isolated from hearts of Antarctic notothenioid fishes that differ in the expression of hemoglobin (Hb) and myoglobin (Mb). Respiration rates were measured at temperatures between 2 and 40°C in Gobionotothen gibberifrons (+Hb/+Mb), Chaenocephalus aceratus (–Hb/–Mb) and Chionodraco rastrospinosus (–Hb/+Mb). Blood osmolarity was measured in all three species and physiological buffers prepared for isolating mitochondria and measuring respiration rates. Respiration rates were higher in mitochondria from G. gibberifrons compared to those from C. aceratus at 2°C, but were similar among all species at temperatures between 10 and 26°C. Respiration rates were significantly lower in icefishes at 35 and 40°C compared to G. gibberifrons. The respiratory control ratio of isolated mitochondria was lower in C. aceratus compared to G. gibberifrons at all temperatures below 35°C. At 35 and 40°C, mitochondria were uncoupled in all species. The Arrhenius break temperature of state III respiration was similar among all three species (30.5 ± 0.9°C) and higher than values previously reported for Antarctic notothenioids, likely due to the higher osmolarity of buffers used in this study. These results suggest that differences in mitochondrial structure, correlated with the expression of oxygen-binding proteins, minimally impact mitochondrial function.     

Temperature dependence of macromolecular interactions in dilute and concentrated hemoglobin solutions

We studied the temperature-dependent effects of intramolecular interactions on the mutual diffusion coefficient of normal human oxygenated hemoglobin in salt solution. We used photon correlation spectroscopy to observe this temperature dependence of the mutual diffusion coefficient of two protein concentrations (1.25 and 17.0 g %) between 13 and 37°C. This coefficient was our probe for monitoring temperature-dependent structural changes of hydrated hemoglobin in solution. Comparison of our measured diffusion coefficient with that predicted by the Stokes-Einstein relationship in terms of solvent or solution viscosity showed a clear transition in the conformation of hemoglobin at approximately 22°C, independent of the hemoglobin concentration. We postulated that at this physiological temperature, a considerable quaternary rearrangement of the hemoglobin chains takes place. We believe this rearrangement changes the effective volume and the hydration sphere of the hemoglobin macromolecule.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Binding of azide ion to methemoglobin at elevated temperatures and the reality of the "compensation" temperature

We have studied the binding of azide ion to ferrihemoglobin at elevated temperatures. Up to a temperature of 45°C there is no difference in the ligand binding behavior of hemoglobin when compared with the results obtained at lower temperatures. The compensation temperature Tc of 290.6 ± 5.3°K, obtained in this study within the temperature range 303–318°K, confirms that the compensation pattern obtained by Lumry and Rajender is not dependent on the temperature range of the experiment but an intrinsic property of the protein conformation.     

Temperature related effects on embryonic development of the Mediterranean locust, Dociostaurus maroccanus

Laboratory studies were conducted to assess the effect of temperature on the development of the eggs of Dociostaurus maroccanus (Thunberg) (Orthoptera, Acrididae) during anatrepsis (stages I–XIV) and during catatrepsis (stages XV–XX). The developmental rates of anatrepsis were studied at five constant temperatures ranging from 10 to 30°C. Egg development occurred over the entire range but at 10°C the embryos were unable to complete anatrepsis. The relationship between temperature and developmental times for completing anatrepsis was analysed by the non‐linear Logan type III model. The optimal temperature estimated for the development of eggs during anatrepsis was 24.7°C; the lower and upper thermal thresholds were 9°C and 31°C, respectively. Once the embryos completed anatrepsis, only those incubated at 15°C continued morphogenesis beyond stage XIV (diapause stage) without a low‐temperature exposure period. The developmental rate of catatrepsis was studied at four constant temperatures ranging from 15°C to 30°C after exposure to low‐temperature, 10°C, for 30, 60 or 90 days. For catatrepsis, temperature and developmental time were linearly and inversely related. Linear regression was used to estimate the lower developmental threshold and the degree days requirements for catatrepsis. Both decreased with longer exposure to the low temperature; the former from 13.8°C to 10.5°C and the latter from 212.8 to 171.5 degree days, following 30 and 90 days at 10°C, respectively. Our results improve the ability of decision support systems for Mediterranean locust pest management by providing better forecasts to land managers and pest advisors.     

The Dynamics of Adaptive Changes in the Spleen of the Hibernating Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

In hibernation season during torpor bouts, the spleen weight and the hemoglobin level, as well as the total and extracted protein contents in the spleen of the ground squirrel Spermophilus undulatus are increased when animals enter torpor and reach maximum values when the body temperature drops below 25°C. All these parameters return to the characteristic values of the euthermic animals during arousal, before the body temperature increases to 20°C. There were no significant differences in the numbers of splenocytes between ground squirrels in interbout euthermia and torpor. The minimum number of splenocytes was observed in animals that entered torpor when the core body temperature was approximately 18°C. The activity of ornithine decarboxylase, a key enzyme in polyamine synthesis, which is correlated with the functional and proliferative status of lymphoid tissue, was the same for the euthermic and summer ground squirrels and decreased monotonically during torpor. Upon arousal of the animals when body temperature was below 29°C, no resumption of the spleen ornithine decarboxylase activity was observed.     

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.     

High temperature acclimation alters upper thermal limits and growth performance of Indian major carp,rohu, Labeo rohita (Hamilton, 1822)

Increase in water temperature due to anthropogenic and climatic changes is expected to affect physiological functions of fish. In this study, we determined high temperature tolerance (CTmax) of a common aquacultured Indian major carp, rohu, Labeo rohita fingerlings (15.96 ± 0.72 g BW, 11.56 ± 0.42 cm TL) followed by acclimatization at three temperatures (30, 33, 36 °C). To determine the CTmax, we analyzed the major hemato-biochemical indices - hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC), blood glucose levels, and erythrocytic nuclear abnormalities (ENAs) and erythrocytic cellular abnormalities (ECAs) of peripheral erythrocytes in the fish sampled at the start and end point at each acclimated temperature. Significantly decreased CTmax of the fish was found at 36 °C compared to 30 °C and 33 °C. The fish in the highest (36 °C) temperature were found with significantly lower Hb and RBC content and significantly higher WBC and blood glucose levels than that of the fishes in the lowest (30 °C) temperature both at the start and end points. The highest frequencies of ENAs and ECAs were found in the highest (36 °C) temperature group compared to the lowest (30 °C) temperature group at both the points. We also evaluated growth performance of the rohu fingerlings reared in the three temperatures for 60 days. The growth parameters - final weight gain, percent weight gain and specific growth rate were the highest at 33 °C and the lowest at 36 °C. The present study revealed that the highest temperature (36 °C) tested here may be hazardous to rohu and the temperature should be kept below 36 °C in the aquaculture setting to avoid physiological damage and growth and production loss to the fish.     

Effect of temperature on functional properties of hemoglobin of crucian carp (<Emphasis Type="Italic">Carassius carassius</Emphasis>)

The effect of temperature on functional properties of the hemoglobin of crucian carp (Carassius carassius) has been studied. It has been revealed that hemoglobin affinity to oxygen increases with increasing temperature (from 12.0 to 33.4°C) at buffer solution pH 6.6 and molarity of 0.05 and 0.005 M. Under impact of temperature, the Bohr effect does not change. Its value also remains constant with increasing molarity of the buffer system from 0.005 to 0.05 M.     

Effect of temperature and pH on the kinetics of methane production, organic nitrogen and phosphorus removal in the batch anaerobic digestion process of cattle manure

A study of the effect of temperature and pH on the kinetics of methane production and organic nitrogen and phosphorus degradation in the anaerobic digestion process of cattle manure was carried out. Two laboratory-scale batch completely mixed reactors, operating at 35v°C (mesophilic temperature), and other two, operating at 60v°C (thermophilic temperature) were used. For each temperature selected, the influent pH values were 7.6 (initial pH of the waste used) and 7.0. The apparent kinetic constants of the biomethanization process increased 2.3 times when the initial pH of the influent was increased from 7.0 to 7.6 at mesophilic temperature. The values found at thermophilic temperature were similar. The kinetic constants of methane production decreased 2.6 and 7.2 times when the operating temperature increased from 35 °C to 60v°C for the experiments carried out at initial pH of 7.0 and 7.6, respectively. The methane yield coefficient (l CH₄ STP/g VS removed) also decreased when the temperature increased from 35v°C to 60v°C for the two initial pH values studied. This behaviour agreed with the major inhibition level observed at thermophilic temperature as a result of the higher organic nitrogen removal and ammonia nitrogen production observed at 60v°C. Specifically, the specific rate constants for organic nitrogen removal and ammonia nitrogen production increased 3.6 and 12 times when the temperature was increased from 35v°C to 60v°C for the experiments carried out at initial pH values of 7.0 and 7.6, respectively. In the same way, the values of the kinetic constant for phosphorus removal were 44% and 80% higher than those obtained at 35v°C for the two initial pH values above-mentioned, respectively. Finally, the experimental values of organic nitrogen and phosphorus concentrations were reproduced with deviations equal to or less than 10% and 15% in every case, respectively.     

Effects of temperature, feeding position and crop growth stage on the population dynamics of the rose grain aphid, Metopolophium dirhodum (Hemiptera: Aphididae)

The population dynamics of Metopolophium dirhodum were studied on winter wheat seedlings at constant (10°C, 15°C, 20°C, 25°C and 30°C) and fluctuating (12(night)-22(day)°C) temperatures, and during booting to early inflorescence, and anthesis to early milky ripe stage, at 19°C. The pre-reproductive development time was decreased by increasing the temperature from 10°C to 25°C. It was significantly shorter when the aphids were feeding during booting to early inflorescence than during anthesis to early milky ripe stage but was similar when the aphids were feeding on the flag, second or third leaves. The total number of nymphs produced/apterous adult was not significantly affected by temperature from 10°C to 25°C but adult reproductive lifespan was reduced by increasing temperature from 10°C and 12–22°C to 15°C, 20°C and 25°C. The daily intrinsic rate of increase changed from 0.11 to 0.25, and the cohort generation time decreased from 31 to 12 days, with increase of temperature from 10°C to 25°C. Reproductive rate was similar when the aphids were feeding on the flag, second or third leaves during booting to early inflorescence at 19°C. The reproductive rate was higher when the aphids fed from mid-inflorescence to mid-milky ripe stage than from mid-milky to early ripe stage. These results were compared with those from other studies. Predictions from a simulation model using development and reproductive rates from this study and literature were compared and the former rates resulted in a more accurate prediction of field observations in 1979, an outbreak year.     

The influence of the thermal environment and other early life events on growth rate of piglets during lactation

The effects of early life events on average daily weight gain from birth to day 21 (ADG) of suckling pigs kept at different room temperatures (15°C, 20°C and 25°C) from birth to weaning were investigated. Data were collected from litters born by 61 sows in a loose housing system. The ADG for piglets with low birth weight (estimated for birth weights below the 10% percentile) was estimated to be 20 to 30 g higher per day at room temperature 20°C to 25°C compared with 15°C. In contrast, the ADG during the lactation period decreased for larger piglets (estimated for birth weights above the 10% percentile) by 28 g/day at room temperature 25°C compared with 15°C. Thus, high ambient temperatures (20°C to 25°C) are favourable for the growth in smaller piglets during lactation. Neither latency to first suckle nor birth-induced hypoxia, measured as concentration of umbilical cord lactate, affected the growth rate of the piglets. Lowest rectal temperature during the first 24 h after birth had a long-term negative effect on ADG (P<0.05), so that piglets with a lowest rectal temperature of 32.8°C (10% percentile) had an ADG which was on average 19 g lower per day than piglets with a rectal temperature of 37.3°C (90% percentile). Our results showed that hypothermia at birth, low birth weight and high number of suckling piglets lead to reduced ADG during the suckling period. The results suggest that keeping the room temperature at 20°C during lactation to some extent could compensate for the otherwise negative effects of low birth weight on ADG in piglets without decreasing the ADG of high birth weight piglets. However, to avoid hypothermia in the smallest piglets it may be beneficial to increase the room temperature above 20°C during the farrowing period of loose housed sows.     

The critical thermal limits for the bullhead, Cottus gobio, from three populations in north-west England

1. The objective was to determine the thermal limits for feeding and survival in the bullhead, Cottus gobio, using juveniles (total length 20–30 mm, live weight 0.5–1.5 g) from one population and adults (50–70 mm, 3.5–5.5 g) from three populations. 2. Fish were acclimated to constant temperatures (3, 7, 10, 15, 20, 25 or 27 °C) and the temperature was then changed at a rate of 1 °C /30 min to determine the critical limits for feeding, survival over 7 days (incipient lethal temperature), or survival for 10 min or less (ultimate lethal temperature). The rate of 1 °C/30 min was the optimum value from preliminary experiments, using nine rates from 0.5 °C/48 h to 18 °C h^?1. As values for adults were not significantly different between populations, they were pooled to provide arithmetic means (with 95% CL) for the thermal limits at each acclimation temperature. 3. Feeding limits increased with acclimation temperature to upper and lower mean values (± 95% CL) of 26.5 ± 0.16 °C and 4.2 ± 0.20 °C for adults, 26.6 ± 0.59 °C and 5.0 ± 0.55 °C for juveniles. Incipient lethal levels defined a tolerance zone within which fish survive indefinitely; upper limits increased with acclimation temperature to a plateau of 27.6 ± 0.22 °C for adults and 27.5 ± 0.47 °C for juveniles, lower limits increased from near 0 °C to 2.5 ± 0.31 °C for adults and 2.7 ± 0.47 °C for juveniles. Ultimate lethal levels increased with acclimation temperature to a plateau of 32.5 ± 0.24 °C for adults and 32.6 ± 0.46 °C for juveniles, whilst the lower limits increased from near 0 to 0.9 ± 0.29 °C. Upper feeding, incipient and ultimate lethal values were significantly lower for juveniles than those for adults at acclimation temperatures < 20, < 20 and < 15 °C, respectively. 4. The thermal tolerance of bullheads was slightly lower than that of stone loach, similar to that of juvenile Atlantic salmon and higher than that of brown trout; the thermal limits for feeding were much wider than those for salmon or trout.     

Effects of cold stress on juvenile Piaractus mesopotamicus and the mitigation by β-carotene

This study investigated the effects of cold stress on morphometrical and hematological biomarkers, energy metabolism, and oxidative stress in different tissues of P. mesopotamicus, and the protective role of β-carotene. Fish were fed with a control diet (CD) and the same diet supplemented with 105 mg/kg β-carotene (BD) for 60 days. After the feeding trial, fish fed CD or BD diets were exposed to control (24 °C) and low temperature (14 °C) for 24 h. Fish (CD and BD) exposed to thermal stress showed lower hepatosomatic index. The hemoglobin increased only in CD-fed fish exposed to 14 °C. Increased glycemia, plasmatic protein depletion, and decreased hepatic glycogen were observed in fish fed the CD, while only the lipid levels in liver were augmented in BD-fed fish exposed at 14 °C. Regarding the oxidative stress, increased antioxidant enzymes activity and lipid peroxidation were observed in CD-fed fish exposed to cold. The two-way ANOVA showed an interaction between dietary treatment and temperature for glucose and oxidative stress biomarkers, with the highest values recorded in 14 °C-exposed fish fed with the CD. Our study demonstrated that cold stress had the greatest impact on fish oxidative status, and β-carotene reduces harmful effects induced by cold in P. mesopotamicus.     

Increasing temperature decreases the predatory effect of the intertidal shanny Lipophrys pholis on an amphipod prey

Interactions between Lipophrys pholis and its amphipod prey Echinogammarus marinus were used to investigate the effect of changing water temperatures, comparing current and predicted mean summer temperatures. Contrary to expectations, predator attack rates significantly decreased with increasing temperature. Handling times were significantly longer at 19° C than at 17 and 15° C and the maximum feeding estimate was significantly lower at 19° C than at 17° C. Functional‐response type changed from a destabilizing type II to the more stabilizing type III with a temperature increase to 19° C. This suggests that a temperature increase can mediate refuge for prey at low densities. Predatory pressure by teleosts may be dampened by a large increase in temperature (here from 15 to 19° C), but a short‐term and smaller temperature increase (to 17° C) may increase destabilizing resource consumption due to high maximum feeding rates; this has implications for the stability of important intertidal ecosystems during warming events.