Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation

The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation.The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS³ concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol.Gelation is not accompanied by a detectable volume change (limits 5 × 10^?5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm³/mol when the aggregates represent one half of the HbS available for polymerization.     

Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues

The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide-sensitive tissue-the germinating pea seedling, and a cyanide-insensitive tissue-the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp decline in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present.     

Separate roles of the latent and infectious periods in shaping the relation between the basic reproduction number and the intrinsic growth rate of infectious disease outbreaks

Two approximations are commonly used to describe the spread of an infectious disease at its early phase: (i) the branching processes based on the generation concept and (ii) the exponential growth over calendar time. The former is characterized by a mean parameter: the reproduction number R₀. The latter is characterized by a growth rate ρ, also known as the Malthusian number. It is common to use empirically observed ρ to assess R₀ using formulae derived either when both the latent and infectious periods follow exponential distributions or assuming both are fixed non-random quantities. This paper first points out that most of these formulae are special cases when the latent and infectious periods are gamma distributed, given by a closed-form solution in Anderson and Watson [1980. On the spread of a disease with gamma distributed latent and infectious periods. Biometrika 67 (1), 191-198]. A more general result will be then established which takes the result in Anderson and Watson [1980. On the spread of a disease with gamma distributed latent and infectious periods. Biometrika 67 (1), 191-198] as its special case. Three aspects separately shape the relationship between ρ and R₀. They are: (i) the intensity of infectious contacts as a counting process; (ii) the distribution of the latent period and (iii) the distribution of the infectious period. This article also distinguishes the generation time from the transmission interval. It shows that whereas the distribution of the generation time can be derived by the latent and infectious period distributions, the distribution of the transmission interval is also determined by the intensity of infectious contacts as a counting process and hence by R₀. Some syntheses among R₀, ρ and the average transmission interval are discussed. Numerical examples and simulation results are supplied to support the theoretical arguments.     

Comparison of Propylene-induced Responses of Immature Fruit of Normal and rin Mutant Tomatoes

Continuous application of propylene to 40 to 80% mature fruits of normal tomato strains (Lycopersicon esculentum Mill.) advanced ripening in fruits of all ages by at least 50%. Although preclimacteric respiration was stimulated by propylene treatment, there was no concomitant increase in ethylene production. Once ripening commenced, the rates of endogenous ethylene production were similar in both propylene-treated and untreated fruits. Continuous exposure to propylene also stimulated respiration in immature fruits of rin, a nonripening mutant. Although respiration reached rates similar to those during the climacteric of comparable normal fruits there was no change in endogenous ethylene production which remained at a low level. Internal ethylene concentrations in attached 45 to 75% mature fruits of rin and a normal strain were similar. It is suggested that the onset of ripening in normal tomato fruit is not controlled by endogenous ethylene, although increased ethylene production is probably an integral part of the ripening processes.     

Sub-cellular damage by copper in the cnidarian Zoanthus robustus

Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110 µg Cu L⁻¹). A decrease in net photosynthesis was observed only at the highest copper concentration (156 µg Cu L⁻¹). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae.     

Circadian and circatidal rhythms of oxygen consumption in the sandy-beach isopod Tylos granulatus Krauss

The sandy-beach isopod Tylos granulatus Krauss burrows at the high tide mark, and has rhythms of nocturnal emergence coincident with the low tide period. Measurements of the respiration rate show that a low rate of oxygen consumption is maintained throughout the day but there is a circatidal rhythmic increase of between 300 and 700% during the nocturnal low tide. The height of this peak and the percentage of animals displaying a peak both increase from spring tide to neap tide suggesting a semi-lunar rhythm. The respiratory peaks have a 24.8 h periodicity, being later each night until low tide falls in the dawn, when there is a ‘switch-back’ so that peaks are then during the subsequent evening low tide. These respiratory rhythms are persistent under constant light and coincide with previously described activity rhythms. The respiratory rhythms considerably reduce metabolic energy losses, particularly as the activity rhythms ensure avoidance of high diurnal temperatures and activity during the cooler nocturnal period.     

ORGANIC MERCURIAL ENCEPHALOPATHY: IN VIVO AND IN VITRO EFFECTS OF METHYL MERCURY ON SYNAPTOSOMAL RESPIRATION

—A reproducible model of subacute methyl mercury (MeHg) intoxication was developed in the adult rat following the daily intragastric administration of 10 mg methyl mercury/kg body wt. Synaptosomes isolated from animals during the latent phase of mercury neurotoxicity (6-10 days) demonstrated no significant change in respiratory control, State 3, State 4, or 2,4-dinitrophenol stimulated respiration with succinate, glutamate or pyruvate plus malate. During the neurotoxic phase, a significant decline in respiratory control was evident with all substrates. Cerebellar synaptosomes revealed qualitatively similar but quantitatively greater inhibition of 2,4-dinitrophenol stimulated respiration during the latent and neurotoxic phases with glutamate. In vitro studies of synaptosome respiration, oxidative phosphorylation and respiratory control with 5-15 μm -methyl mercury revealed a stimulation of initial State 4 respiration, loss of RCI, inhibition of State 3 but no change in the gramicidin or 2,4-dinitrophenol uncoupled rate supported by pyruvate-malate. Phosphate did not relieve the State 3 inhibition. At 25 μm -methyl mercury and above, considerable inhibition of electron transfer occurred. At this concentration, cytochrome c oxidase was inhibited 50%. Isosmotic replacement of medium KC1 by mannitol reduced the MeHg stimulation of State 4 respiration but had no effect on MeHg inhibition of ADP stimulated respiration. Half-maximal stimulation of State 4 respiration by MeHg occurred at [K]⁺⋍ 6 mm . These findings are compatible with an energy-linked methyl mercury induced cation translocation across the synaptosome (mitochondrial) membrane.     

GEOTROPIC EXCITATION IN HELIX

Rotation of an inclined surface on which Helix is creeping straight upward, such that the axis of the animal is turned at a right angle to its previous position, but in the same plane, leads to negatively geotropic orientation after a measurable latent period or reaction time. The duration of the latent period is a function of the slope of the surface. The magnitude of the standard deviation of the mean latent period is directly proportional to the mean latent period itself, so that the relative variability of response is constant. The dependence of reaction time upon extent of displacement from symmetrical orientation in the gravitational field is found also by tilting the supporting surface, without rotation in the animal''s own plane. On slopes up to 55°, the relation between latent period and the sine of the slope is hyperbolic; above this inclination, the latent period sharply declines. This change in the curve is not affected by the attachment of moderate loads to the snail''s shell (up to 1/3 of its own mass), and is probably a consequence of loss of passive stable equilibrium when rotated. When added loads do not too greatly extend the snail''s anterior musculature, the latent period for the geotropic reaction is decreased, and, proportionately, its σ. These facts are discussed from the standpoint that geotropic excitation in these gasteropods is due to impressed muscle-tensions.     

An Asymmetric Respiratory Response Occurring when Fluoride and Copper Ions are Applied Jointly to Chlorella vulgaris

When a cell suspension of Chlorella vulgaris is treated with a mixture of copper and fluoride ions, the respiration almost ceases even though the two ions individually have lit t le inhibitory effect. If instead of adding the ions simultaneously, the cells are subjected to a period of pretreatment with copper sulphate before sodium fluoride the inhibition of respiration becomes more severe as the pretreatment time is lengthened, except at copper levels so high that inhibition is almost complete at all lengths of pretreatment. When, however, pretreatment is with fluoride ions, the inhibition becomes less as the pretreatment time is lengthened, eventually disappearing when the time interval is 90 min or more. Measurements of the amount of copper in the cells indicate that this asymmetry of response cannot be ascribed to differences in the rate at which copper is taken up. It is suggested that fluoride blocks the main respiratory pathway and that copper probably blocks the hexose monophosphate shunt.     

Water Content and Respiration Rate of Bean Cotyledons

The course of water uptake and respiration rate rise in cotyledonsof Phaseolus vulgaris is divided into three phases. In the first phase lasting 10–16 hrs. respiration rateis controlled by water content; desiccation and reimbibitioninfluence cotyledon water content and respiration rate alikeand the changes are reversible; low temperature prevents watercontent rising above 50 per cent. and also limits respirationrate; both processes have Q₁₀ near unity. The second phase lasting 3–8 hrs. is characterized bya pause in both water uptake and respiration rate rise. In the third phase respiration rate continues to rise untilthe fifth day, after which it falls steadily until eventuallythe cotyledons absciss. The period of rising respiration isone of metabolic activity, the rise having a high Q₁₀ and beingprevented by low temperature. Desiccation at this stage is irreversible.     

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl₄ administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V₃) and 4 (V₄) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V₃ and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl₄ resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).     

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.     

A STUDY OF THE CIRCULATION,BLOOD PRESSURE,AND RESPIRATION OF SHARKS

The average branchial blood pressure in sand sharks was 32 mm. of mercury. The highest recorded in a resting animal was 43 mm. The average dorsal or systemic pressure was 23.3 mm.; highest 30 mm. The ratio of branchial to systemic pressure is about 3 to 2. The pressure in both systems keeps up well under trauma; but under experimental conditions, with or without manipulation of viscera, slowly falls after several hours. It rises with muscular effort, and a long rise usually follows stoppage of struggling. It rises when sodium carbonate is injected. The adrenalin curve resembles that in a mammal. Spontaneous rises and falls not attributable to the heart occur. Light in some animals increases blood pressure. It is suspected that these fishes have a vasomotor apparatus. The heart rate except after trauma is practically always the same as the respiration rate, and there is some reason for believing that the heart rate is determined by the respiration rate. When not in step with respiration, the heart is slower and often in a simple ratio with respiration. The heart is inhibited by all sorts of stimuli applied practically anywhere (except to the liver?). This effect is abolished by atropin. Respiration is faster in small animals and averages 24 per minute. Respiration slowly decreases in strength with little change in rate. Usually respiration ceases long before the heart stops.     

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl₂, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl₂, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl₂. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl₂. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.     

Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration

In spinach (Spinacia oleracea Hybrid 102 [New World seeds]) and wheat (Triticum aestivum L. cv Gabo) leaves, O₂ uptake rates in the dark were faster after the plants had been allowed to photosynthesize for a period of several hours. Alternative path activity also increased following a period of photosynthesis in these leaves. No such effects were observed with isolated mitochondria. In spinach and wheat leaves, the level of fructose plus glucose decreased during a period of darkness. In pea (Pisum sativum cv Alaska) leaves, the level of these sugars did not vary significantly during the day, and respiratory rates were also constant. In slices cut from wheat leaves harvested at the end of the night, addition of sugars increased the rate of respiration and engaged the previously latent alternative oxidase. In pea leaves, O₂ uptake in the first few minutes following illumination was faster than that observed before illumination, but declined during the next 15 to 20 minutes. Adding the alternative oxidase inhibitor salicylhydroxamic acid, or imposing high bicarbonate concentrations during the period of photosynthesis, prevented the rise in O₂ uptake rate during the immediate post illumination period.

We conclude that the level of respiratory substrate in leaves determines their rate of O₂ uptake, and the degree to which the alternative path contributes to that O₂ uptake.

     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.     

Respiration in Branchinella Kugenumaensis (Crustacea-Anostraca) inhabiting tropical astatic ponds

The oxygen consumption (routine rate) of Branchinella kugenumaensis, inhabiting tropical astatic ponds is measured at 28°C. Two distinct trends are observed in the oxygen consumption by this fairy shrimp; young, immature animals have a higher rate than the older, sexually mature animals. Branchinella kugenumaensis falls under the intermediate group of Bertalanffy, where respiration falls between the respiration surface proportional and the respiration weight proportional metabolism.     

Effects of CO(2) Enrichment and Carbohydrate Content on the Dark Respiration of Soybeans

During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO₂ per liter air, was 1.45 milligrams CO₂ evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO₂ per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO₂ enriched compared with CO₂ normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO₂ enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO₂ enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO₂ adapted plants, but not in the control plants.

Mitochondria, isolated simultaneously from the leaves of CO₂ enriched and control plants, showed no difference in NADH or malate-glutamate dependent O₂ uptake, and there were no observed differences in the specific activities of NAD⁺ linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O₂ uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO₂ enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status.

     

The course of respiration during the life cycle of Chlorella cells

Endogenous and glucose respiration were studied during the life history of Chlorella pyrenoidosa. A generalized picture of the course of respiration during the life cycle is suggested. At the liberation of daughter cells from the wall of the mother cell, or soon after, the respiration rate reaches its lowest level. If the daughter cells are placed in light the respiration rate rapidly increases with time, soon reaches a maximum, and then declines slowly. Two factors are important in the initial increase—the early developmental stage of the cells and the influence of light. In autotrophically developing algae the parts played by developmental processes and by light have not been separated. Direct activation of respiratory enzymes by light, in addition to the level of respiratory substrate, cannot be excluded. The decline of respiration rate over most of the cell history seems to have no connection with light and is probably bound to the developmental processes per se. Darkening the suspension interrupts growth and induces liberation of daughter cells, with concomitant faster decrease in respiration rate. The rate of respiration of small daughter cells decreases in darkness only slowly with time. Illumination seems necessary to bring these cells back to a high level of respiratory activity.     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.