Interrelationships between water and cellular metabolism in Artemia cysts. XI. Density measurements

Cysts of the crustacean Artemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (rho w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (rho c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (Wf), and temperature dependence was measured for 0.61 Wf over 2-41 degrees C. The following refer to 25 degrees C. No marked change was detected in rho c until the water content exceeded 0.15 Wf, at which rho c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function of Wf. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.     

Interrelationships between water and cellular metabolism in Artemia cysts VII. Free amino acids

The concentration of total ninhydrin-positive material (NPM) soluble in 5% trichloroacetic acid was measured in cysts of the brine shrimp, Artemia salina, as a function of hydration level. No net change in NPM was observed until the cysts had achieved a water content of about 0.65 g H₂O/g of initially dry cysts. Above this hydration threshold the NPM content increased markedly. Examination of the free amino acid composition of cysts incubated at selected hydration levels revealed that almost all of the amino acids underwent net change above the hydration threshold. However, just below this threshold, the free amino acid composition was essentially the same as in fully dried cysts. The activity generating net changes in the concentration of free amino acids above the hydration threshold was shown to be metabolic in nature and restricted to the cellular component of the cyst.     

Interrelationships between water and cellular metabolism in Artemia cysts. VI. RNA and protein synthesis

Using ¹⁴CO₂ as a labelled precursor the relationship between the initiation of protein and RNA synthesis, and water concentration, has been examined in cysts (encysted embryos) of the brine shrimp, Artemia salina. Although incorporation of radioactivity into amino acids and nucleotides occurred in cysts at hydrations as low as 0.3 g H₂O/g dried cysts, incorporation into proteins and RNA was not measurable until the cysts had achieved a hydration in the range of 0.6–0.6 g/g. In no case was radioactivity detected in DNA of unemerged cysts. Fully hydrated cysts (about 1.3 g/g) that were actively synthesizing proteins and RNA, stopped doing so when dehydrated to levels below the same hydration range: thus, the hydration dependence does not involve appreciable hysteresis. The hydration range required to initiate synthesis of these macromolecules is essentially the same as that previously shown to initiate embryonic development.     

Interrelationships between water and cellular metabolism in Artemia cysts. IV. Adenosine 5'-triphosphate and cyst hydration.

The concentration of ATP in cysts of the brine shrimp, Artemia salina, has been studied as a function of cyst hydration. Cysts dried over CaSO4 contain 0.02 gH2O/g cysts and 0.54 +/- 0.05 (S.D.) mumoles of ATP/g dried cysts. Addition of water up to 0.05 g/g cysts produced no net change in the level of ATP during incubation. Hydration levels between 0.05 and 0.62 g/g cysts resulted in a net loss of ATP, whereas above 0.65 g/g cysts a net increase was observed with incubation time. No net change in the amount of ATP, compared with dried cysts, was detected between the latter two hydrations. These results, when integrated with those from previous work, indicate that conventional aerobic energy metabolism does not begin until cyst hydrations of about 0.65 g/g are achieved. The fate of ATP in cysts hydrated to levels lower than 0.65 g/g was discussed.     

Interrelationships between water and cellular metabolism inArtemia cysts

Cysts of the crustaceanArtemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (ρ_w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (ρ_c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (W _f), and temperature dependence was measured for 0.61W _f over 2–41°C. The following refer to 25°C. No marked change was detected in ρ_c until the water content exceeded 0.15W _f, at which ρ_c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function ofW _f. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

Interrelationships between water and cell metabolism in Artemia cysts X. Microwave dielectric studies

Artemia cysts are composed of an inner mass of about 4000 cells surrounded by an acellular shell. This system can undergo cycles of hydration-dehydration without viability loss, and is a useful model for the study of intracellular water. We have measured the relative permittivity (ε′) of these cysts as a function of water content over the frequency range 0.8–70 GHz. Detailed analysis of the data for cysts containing close to 1 g H₂O/g dry weight indicates that a significant fraction of the total water in this system exhibits dielectric behavior different from that of pure water: the distribution parameter (α) for the dispersion analyzed by the Cole-Cole equation deviates from zero, and the permittivity of cyst water appears to be significantly lower than that of pure liquid.     

Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation.

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.     

Cryptic form of mRNA in dormant Artemia salina cysts.

Dormant $\text{Artemia salina}$ cysts are almost devoid of polysomal structures but contain appreciable quantities of mRNA that sediments mainly as a 40S complex in sucrose gradients. The mRNA can be isolated from this complex and efficiently translated in a wheat germ cell-free system, although the 40S complex itself is inactive. During rehydration of the cysts, mRNA becomes increasingly involved in polysomal complexes which can be actively translated in the cell-free system.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.II=Jessup and Fowler, 1976 b     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

Summary The effect of the nature and concentration of the nitrogen source on respiratory activity and removal of carbohydrate from the medium in suspension cultured sycamore (Acer pseudoplatanus L.) cells was determined. Comparison was also made of the rates of uptake of the two alternative nitrogen sources, nitrate and glutamate, at differing initial nitrogen concentrations within the range 7–14 mM. The initial pH of the culture medium before inoculation was 5.2; after inoculation the pH of both nitrate and glutamate cultures rose to reach an eventual level in the range 7.0–7.1. Glutamate was removed from the medium more slowly than nitrate. Under the particular conditions of culture used the growth of the cells was nitrogen limited. Sugar uptake from the medium continued for some time after the nitrogen in the medium was depleted. The data show that although cell division and protein content are nitrogen-limited, dry weight and fresh weight yields may also be determined in a complex interaction through carbohydrate availability. There were no obvious differences in respiratory activity between cultures grown on nitrate or glutamate.     

Interrelationships between the enzymes of ethanolamine metabolism in Escherichia coli

The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen. Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12. Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol. wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol. wts 520000 and 370000) in the presence of both co-inducers. A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase. No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control. Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E. coli.     

Interrelationships between egg mass and adult body mass and metabolism among passerine birds

Summary The regression of egg mass of Passeriformes against female body mass (n=1244) and basal metabolic rate of Passeriformes against body mass (n=159) have similar slopes, indicating that for this group egg mass is directly proportional to basal metabolic rate and that the relative egg mass (% of body mass) is directly proportional to the weight-specific metabolism. This relationship and the average caloric density of passerine eggs (n=38) provides an estimate of the cost of egg production, which for single eggs is 41 % of the basal metabolic rate.

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Beziehungen zwischen Eigewicht, Körpergewicht und Stoffwechsel bei Sperlingsvögeln

Zusammenfassung Die Regressionsgeraden von Eigewichten der Passeriformes und weiblicher Körpergewichte (N=1244) einerseits sowie von Ruheumsatz und Körpergewichten (N=159) andererseits haben vergleichbare Steigungen. Das bedeutet, (1) daß für diese Vogelordnung das Eigewicht proportional zum Ruheumsatz und (2) daß das relative Eigewicht (% des Körpergewichtes) proportional zum gewichtspezifischen Stoffwechsel ist. Dieses Verhältnis und der durchschnittliche kalorische Wert der Eier (N=38) erlauben die Kosten der Eiproduktion zu schätzen: sie betragen für das einzelne Ei 41 % des Ruheumsatzes.

     

Assay for nanogram quantities of DNA in cellular homogenates.

The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.