The temperature jump relaxation technique.

The temperature jump relaxation technique is a convenient and general means of studying rapid reversible reactions of biological macromolecules. Recent advances in automatic data acquisition and the introduction of different optical detection systems will soon allow us to exploit the full potential of kinetic measurements near equilibrium. On the other hand, the kinetic approach can be quite limited if not accompanied by detailed structural and thermodynamic studies. Finally, we must accept the fact that one can rarely demonstrate a reaction mechanism to the exclusion of all plausible alternative models.     

Effect of water potential on sol-gel transition and intermolecular interaction of gelatin near the transition temperature

The sol-gel transition of gelatin, measured by thermal analysis and viscosity measurement, was analyzed in terms of the change in hydration state of polymer molecules. A new thermodynamic model was proposed in which the effect of water potential is explicitly taken into account for the evaluation of the free energy change in the sol-gel transition process. Because of the large number of water molecules involved and the small free energy change in the transition process, the contribution of water activity, a(W), was proved to be not negligible in the sol-gel transition process in solutions containing such low-molecular cosolutes as sugars, glycerol, urea, and formamide. The gel-stabilization effect of sugars and glycerol was linear with a(W), which seemed consistent with the contribution of water potential in the proposed model. The different stabilization effect among sugars and glycerol was explained by the difference in solvent ordering, which affects hydrophobic interaction among protein molecules. The gel-destabilization effect of urea and formamide could be explained only by the direct binding of them to protein molecules through hydrogen bonding. On the contrary, the polymer-polymer interaction, measured by the viscosity analysis, in polyethyleneglycol and dextran solutions was not sensitive to the change in a(W), suggesting that no substantial change in hydration state with a(W) occurred in these polymer solutions.     

The influence of storage temperature on the transition, activation enthalpy, and activity of enzymes associated with inner mitochondrial membranes

The effects of storage at low temperature on the transition in enzyme function, Tf*, and the Arrhenius activation energy, Ea, were determined for several enzymes associated with the inner membrane of rat liver mitochondria. The enzymes studied were succinate:cytochrome c reductase, cytochrome c oxidase, beta-hydroxybutyrate dehydrogenase, and oligomycin-sensitive, Mg2+-activated ATPase. For freshly isolated mitochondria the Tf*, for succinate:cytochrome c reductase and cytochrome c oxidase, occurred at approximately 23 degrees C and was coincident with a transition in structure, Ts*, determined as the change in temperature coefficient of motion for a spin label intercalated with the membrane lipids. This suggest that the change in thermal response of the membrane-associated enzymes is related to a change in molecular ordering of the membrane lipids. When mitochondria were stored at -12 degrees C, the specific activities of succinate:cytochrome c reductase and cytochrome c oxidase decreased. Concomitant with these changes the Ea, above Tf*, increased. After 100 days storage at -12 degrees C, Ea above Tf* approached the value for Ea below Tf* such that the transition in thermal response could no longer be detected. In contrast, for mitochondria stored at -196 degrees C, although the specific activity declined over the 100 days storage, no changes in either Ea or Tf* were evident. The results indicate a need for caution in evaluating comparative studies of Tf and Ea, for membrane-associated enzymes, using mitochondria which have been frozen and stored.     

Dissociation of single-stranded DNA from nucleosomes following modification with acetic anhydride

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic strength.     

In defence of the high energy phosphate bond

The concept of the “high energy phosphate bond” has recently been strongly criticised by Banks &; Vernon. The criticisms were: (i) energy cannot be stored in molecules; let alone in bonds; (ii) a muscle is an open system, so that the free energy of hydrolysis of ATP is irrelevant; (iii) biological reactions cannot be at equilibrium, therefore they must be inefficient. It is argued that all of these criticisms arise because the timescale appropriate to molecular events has been left out; in particular “stored energy” needs to be defined relative to the machine which uses that energy. The criticisms may be answered using a conceptual framework which overcomes this deficiency and which was developed previously to extend classical thermodynamics to the molecular level. Criticism (i) is refuted by discussing in detail the chemiluminescent reaction of rubrene oxide, which can only be described as liberating internal energy stored in a single molecule; while it is true that entropy cannot be retained in a single molecule for long enough to do useful work with it, there is nothing which forbids internal energy from being so stored. Criticism (ii) would be true if, and only if, a muscle uses the same kind of mechanism as do the ordinary chemical machines with which we are familiar (batteries, etc). But this assumption is shown to be false; a muscle cannot use this type of mechanism. Criticism (iii) is answered by finding the conditions under which a biological machine could in fact approach 100% efficiency. These conditions are: (i) in metabolism ΔF = 0; the steps are in thermal equilibrium; (ii) in the molecular machines themselves ΔS = 0, so that ΔF = ΔH; the machines are mechanical. It is also shown that not only are these conditions perfectly conceivable, but also that there is good evidence that, over the course of evolution, they have actually been attained.     

Effects of extreme,fluctuating temperature events on life history traits of the grain aphid,Sitobion avenae

Altered temperatures affect insects’ life history traits, such as development period and fecundity, which ultimately determine population growth rates. Understanding insects’ thermal biology is therefore integral to population forecasting and pest management decision‐making such as when to utilise crop spraying or biological control. Aphids are important crop pests in temperate regions, causing considerable yield losses. The aphid thermal‐biology literature is, however, heavily biased towards the effects of rising mean temperatures, whereas the effects of fluctuating, extreme climatic events (e.g., heat waves and sub‐zero cold periods) are largely overlooked. This study assessed the effects of laboratory‐simulated heat waves and sub‐zero cold periods on the survival, development period, and fecundity of the grain aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae: Microsiphini), in addition to assessing maternal effects on the birth weight and development period of the offspring of exposed individuals. Exposure to heat stress periods (total of 16 h at 30 °C) significantly reduced aphid fecundity and increased physiological development period (in day‐degrees) resulting in a reduced population growth rate. Cold exposure (total of 1.33 h at ?15 °C) reduced population growth rate due to an elongated development period (in days), but did not affect fecundity or physiological development period (in day‐degrees). Both cold and heat stress significantly reduced aphid survival. Maternal experience of heat stress reduced nymphal birth weight although nymphal development period was not affected by either cold or heat stress. The results suggest that including the effects of fluctuating, extreme temperature events on aphid life history in population forecast models is likely to be of great importance to pest management decision‐making. The demonstration of maternal effects on birth weight also suggests that cross‐generational effects of heat waves on population growth rates could occur.     

HEMOLYTIC ANEMIAS,Recent Advances in Diagnosis and Treatment

The hemolytic anemias of unknown cause can be separated into two main groups: (1) those produced by a defect in cell structure, which is usually hereditary, and (2) those due to a hemolysin of immune-body type.The hemolytic anemias associated with hypersensitivity to drugs and disease processes such as leukemia are less well understood and need further investigation.Splenectomy is the only effective treatment in congenital hemolytic jaundice and in acquired hemolytic anemia; the operation should be carried out promptly in most cases. Transfusion may be used in all varieties of hemolytic disease and is the only effective form of therapy in sickle-cell anemia and paroxysmal nocturnal hemoglobinuria.     

Acid-base Balance in Acute Gastrointestinal Bleeding

Acid-base balance has been studied in 21 patients with acute upper gastrointestinal bleeding. A low plasma bicarbonate concentration was found in nine patients, accompanied in each case by a base deficit of more than 3 mEq/litre, indicating a metabolic acidosis. Three patients had a low blood pH. Hyperlactataemia appeared to be a major cause of the acidosis. This was not accompanied by a raised blood pyruvate concentration. The hyperlactataemia could not be accounted for on the basis of hyperventilation, intravenous infusion of dextrose, or arterial hypoxaemia. Before blood transfusion it was most pronounced in patients who were clinically shocked, suggesting that it may have resulted from poor tissue perfusion and anaerobic glycolysis. Blood transfusion resulted in a rise in lactate concentration in seven patients who were not clinically shocked, and failed to reverse a severe uncompensated acidosis in a patient who was clinically shocked. These effects of blood transfusion are probably due to the fact that red blood cells in stored bank blood, with added acid-citrate-dextrose solution, metabolize the dextrose anaerobically to lactic acid. Monitoring of acid-base balance is recommended in patients with acute gastrointestinal bleeding who are clinically shocked. A metabolic acidosis can then be corrected with intravenous sodium bicarbonate.     

Habitat selection as a source of biological diversity

Summary Question: What are the conditions required for natural selection to produce phenotypes specially adapted to the various habitats available in nature? Model: Assume there are two habitat types and one or two phenotypes of the same or different species. The phenotypes do not recognize any spatial differences among patches of the same habitat type. Possible evolutionary winners can do better in one habitat only by relinquishing some ability in the other. Results: If only one phenotype is present, it will be an intermediate (unless one of the two habitat types is so rare and unproductive that its effects can be ignored by natural selection). Even if two phenotypes are introduced, natural selection should generally restore monomorphism if habitat selection is not ever favored (e.g. if search costs are high). But if search costs and environmental variation are zero, dimorphism can be expected. And if they are small, then although monomorphism is stable, its basin of attraction is small, and invasion by a second form (such as a sibling species) can provide the discontinuous jump needed to put the system in the other basin of attraction. Once there, dimorphic extremism coevolves. Each successful morph is as specialized as possible on one of the habitats. Competition between the morphs is eliminated. Environmental variation may constrict the basin, but once a point is captured by it, the system approaches dimorphic extremism anyway. In general, whatever promotes the behavior of habitat selection also promotes the evolution of extreme morphologies and physiologies.     

The determination of the kinetics of polysaccharide thermal degradation using high temperature viscosity measurements

Information about the thermal degradation of the polysaccharides sodium alginate, carrageenan and carboxymethyl cellulose has been obtained from the time dependence of the viscosity at high temperatures measured using a slit viscometer. The viscosity is related to the molecular weight using previously-published relations between the zero shear specific viscosity and the coil overlap parameter in conjunction with the appropriate Mark-Houwink equation. It is found that alginate is much less stable than carboxymethyl cellulose and carrageenan. Activation energies for depolymerisation obtained from Arrhenius plots in the presence of oxygen ranged from 50 kJ/mol for alginate to 105 kJ/mol for κ-carrageenan.     

Hemolytic anemias recent advances in diagnosis and treatment

THE HEMOLYTIC ANEMIAS OF UNKNOWN CAUSE CAN BE SEPARATED INTO TWO MAIN GROUPS: (1) those produced by a defect in cell structure, which is usually hereditary, and (2) those due to a hemolysin of immune-body type.The hemolytic anemias associated with hypersensitivity to drugs and disease processes such as leukemia are less well understood and need further investigation. Splenectomy is the only effective treatment in congenital hemolytic jaundice and in acquired hemolytic anemia; the operation should be carried out promptly in most cases. Transfusion may be used in all varieties of hemolytic disease and is the only effective form of therapy in sickle-cell anemia and paroxysmal nocturnal hemoglobinuria.     

The energetics of the jump of the locust Schistocerca gregaria.

The anatomy of the metathoracic leg is redescribed with particular reference to storage of energy in cuticular elements and the way in which the stored energy is used in jumping. The jump of adult male locusts requires an energy of 9 mJ and that of the female requires 11 mJ. The semilunar processes of each metafemur store 4 mJ at a stress of 15 N, and the extensor tibiae apodeme stores a further 3 mJ at the same stress. The total stored energy in both metathoracic legs is 14 mJ. The extensor tibiae muscle produces a maximum isometric force of over 15 N at 30 degrees C and, when loaded with the extensor apodeme and semilunar processes, attains this force in 0.3 sec with a strain of 0.8 mm. The peak power output is 36 mW or 0.45 W.g-1. The peak isometric force is attained when the tibia is fully flexed and the force falls as the tibia extends. The extensor tibiae muscle A band is 5.5 mum long and the peak force is over 0.75 N.m-2. The peak velocity of shortening is 7 mm.sec-1 or about 1.75 lengths/sec at 30 degrees C. The tensile strength of the extensor apodeme is 0.6 kN.mm-2 and Young's modulus is 19 kN.mm-2. The safety factor does not exceed 1.2 and the safety factor of the semilunar processes and tibial cuticle is little higher. The jump impulse lasts 25-30 msec. A velocity of 3.2 m.sec-1 is reached after a peak acceleration of 180 m.sec-2. The peak power output is 0.75 W at close to maximum velocity. Energy losses in rotating the femur and tibia are small and it is shown that the leg is able to extend at 7 times the normal rate with losses of about 20%. Most of the stored energy is converted to kinetic energy as the animal jumps. A model is based on the relaxation of a spring that has the properties of the elastic elements of the locust leg into a lever with the same kinematics as the locust leg produces a force-distance curve similar to that measured for locust jumps. The major part of the jump energy is stored before the jump.     

Genotyping of alpha-thalassemia in microcytic hypochromic anemia patients from North India

Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alpha-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha3.7 deletion, -alpha4.2 deletion, Hb Constant Spring, and a-triplications was done with polymerase chain reaction. The overall frequency of -alpha3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for a-thalassemia is 0.09. The subgroup analysis showed that co-inheritance of a-deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as a-thalassemia carriers. The a-gene mutation is quite common in the Indian subcontinent. Molecular genotyping of a-thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.     

Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density

The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.     

The concept of energy height in animal locomotion: separating mechanics from physiology

The distance flown in gliding is proportional to the starting height, not to the starting potential energy, and it is independent of the body mass. By analogy, in powered flight, the quantity of stored fuel can be converted into a virtual "fuel energy height", defined as the height to which the fuel energy could lift the bird against gravity, if it were converted into work. This is a logarithmic function of the fuel fraction, not of the absolute amount of fuel, or of the body mass. It takes account of the strength of gravity, and of the efficiency with which fuel energy is converted into work. The "performance number" is the gradient on which a migrating bird comes "down" from its initial fuel energy height. It is mechanical (not physiological) in character, and corresponds to the lift:drag ratio in a fixed-wing aircraft. The concept of range as an initial energy height multiplied by a performance number can also be applied to swimming and running animals. Performance number, and also the related variable "cost of transport", are both independent of gravity in flying and running, but not in swimming.Migration by thermal soaring is analogous to powered flight with stopovers, except that the bird replenishes its potential energy by climbing in thermals, rather than replenishing fuel energy during stopovers. Rates of climb in thermals are typically higher than fuel energy rates of climb, but the available height band is two orders of magnitude smaller, and the intervals at which energy replenishment is needed are correspondingly shorter. Albatrosses replenish their kinetic energy by exploiting discontinuities in the wind flow over waves, requiring replenishment at intervals of tens of seconds, a further two orders of magnitude shorter than in thermal soaring.Fat energy height can be used as a measure of "condition", which is independent of the size or type of the animal. The fat energy height at which a migrant must arrive on the breeding grounds, in order to breed successfully, reflects the ecological characteristics of the habitat, not the size or character of the bird. Energy height expresses what an animal or machine can do with its stored energy, not the amount of energy.     

Active leg stiffness and energy stored in the muscles during maximal counter movement jump in the aged

The purpose of this study was to examine the effects of age on active leg stiffness adjustment, electromyogram (EMG) activities and energy stored during eccentric and concentric phases in performing a maximal functional task involving stretch-shorten cycle. Ten young (24.3 ± 2 years) and 10 old (68.6 ± 5 years) healthy male subjects were filmed during maximal performance of counter movement jump (CMJ) and squat jump (SJ) on force plate. Integrated EMG (IEMG), ground reaction force (GRF), active leg stiffness, energy stored/returned and active work done by the muscles were compared between two groups on eccentric (ECC) and concentric (CON) phases of CMJ. The GRF, leg stiffness and energy stored in ECC and GRF, IEMG, energy returned and active work in CON were less in the elderly (p < 0.05). These results demonstrate that the neuromuscular function of adjusting active stiffness, storing elastic energy and optimizing the performance may decrease with age during CMJ.     

Quantitative erythrocyte membrane proteome analysis with Blue-Native/SDS PAGE

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient.This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.     

Heterogeneity of sickle-cell anemia based on a profile of hematological variables.

Factors that influence the heterogeneity of the disease expression of sickle-cell anemia are not well understood. This study examines the ability of a profile of six hematological variables (HB, HCT, RBC, %Hb F, MCV, and %HBA2) to predict the severity of disease measured on 225 patients ranging from 0.2 to 18 years of age. Four subgroups of patients were identified separately in each sex using cluster analysis techniques. In each sex, mean hemoglobin concentration and percent Hb F increased across the four clusters from 7 to 10 gm/dl and from 7% to 16%, respectively. Mean cell volumes were approximately 90, 80, 90, and 75 in groups 1, 2, 3, and 4, respectively; thus MCV did not increase in an orderly progression along with HB and %Hb F. We studied the distribution of four anthropometric, five physical examination, and seven clinical measures of disease severity among clusters. In each sex, subgroups differed significantly (P less than .05) for percent ever hospitalized for sickle-cell anemia, percent ever transfused, and percent with bone-age delays greater than 1 year. In addition, male clusters differed significantly for percent ever having had pneumonia, priapism, or dactylitis, and females differed significantly for height and weight. %Hb F and its inverse relationship with %HBA2 was more highly associated with the measures of severity than the degree of anemia or MCV. This study establishes the utility of a vector of hematological variables as a predictor of heterogeneity of measures of clinical manifestations among young patients with sickle-cell anemia. The role of alpha-thalassemia and genetic factors that affect Hb F levels were considered as possible explanations for the observed heterogeneity.     

Tendon elasticity and muscle function

Vertebrate animals exploit the elastic properties of their tendons in several different ways. Firstly, metabolic energy can be saved in locomotion if tendons stretch and then recoil, storing and returning elastic strain energy, as the animal loses and regains kinetic energy. Leg tendons save energy in this way when birds and mammals run, and an aponeurosis in the back is also important in galloping mammals. Tendons may have similar energy-saving roles in other modes of locomotion, for example in cetacean swimming. Secondly, tendons can recoil elastically much faster than muscles can shorten, enabling animals to jump further than they otherwise could. Thirdly, tendon elasticity affects the control of muscles, enhancing force control at the expense of position control.