Intracellular optical physiology of the bee's eye

Summary Intracellular optical physiology is a newly developed, non-invasive technique for recording from single types of insect photoreceptor cells. An intact animal is mounted on the goniometer stage of a double-beam, incident-light microspectrophotometer. The stimulating beam delivers monochromatic flashes that evoke pupillary responses from photoreceptor cells in a localized region of the eye. The measuring beam delivers red or infrared illumination that continuously measures the changes in reflectance that accompany the pupillary response. In this paper on the worker bee we demonstrate two experimental conditions under which only one of the three spectral types of photoreceptor dominates the measured response.When the eye is dark-adapted, and increases in reflectance are measured with an infrared beam, threshold responses evoked by long flashes (40 s or more) are dominated by that cell type which is most sensitive to the stimulus. The following data were taken from the dorsal (ventral) poles of the bee's eye: At 350 nm, a quantum flux of 0.1 (0.05)×10 ¹² photons/cm ²/s/numerical aperture of 0.2 produces a threshold response that originates only from the UV-receptors. At 430 nm, a quantum flux of 0.6 (0.2) units produces a threshold response that is dominated by the blue-receptors. At 530 nm, a flux of 1.0 (0.1) units produces a threshold response that originates only from the green-receptors.When the eye is red-adapted, the pupillary responses to short flashes (10 s or less) are dominated by the green-receptors. Under this condition the pupillary action-spectrum for sensitized green-receptors is the same as the electrophysiological spectral sensitivity function of the minimally coupled green-receptor. This is true throughout the spectral range 350 nm–650 nm (Fig. 3).If the duration of stimulating flashes is increased to 40 s, the shape of the action spectrum is unchanged for wavelengths greater than 470 nm, but is significantly elevated, by as much as 1.8 log-units, for wave-lengths less than 420 nm (Fig. 4). In this case the UV-receptors dominate the pupillary response at short wavelengths, while the green-receptors dominate it at long wavelengths.We used these effects to determine that all three spectral types of receptor are present in the regions of both dorsal and ventral poles, as well as in the frontal region of the eye.This work was supported by grants EY01140 and EY00785 from the National Eye Institute, U.S.P.H.S. (to GDB), by the Connecticut Lions Eye Research Foundation (to GDB), and by a grant from the University of Zurich (to RW), and by grant 3.529.075 from the Swiss National Science Foundation (to RW) including a Senior Research Fellowship awarded to GDB. We thank Dr. Thomas Labhart for many helpful discussions and for allowing us to refer to his unpublished data, and Dr. Doekele Stavenga for his critical, constructive comments.     

Intracellular Organization: Evolutionary Origins and Possible Consequences to Metabolic Rate Control in Vertebrates

We adopt the position that metabolism originated at (or near)mineral surfaces prior to the origin of the first cells. Basedon current views of the organization of contemporary animalcells we speculate that the metabolism of the immediate ancestorsof eukaryotic cells required these non-biological surfaces,but that the latter were subsequently replaced by membranes,and nuclear and cytoplasmic matrix proteins which, we argue,remain as required participants in the intermediary metabolismof contemporary eukaryotic cells. The idea that such an lntracellularorganization could have provided a fundamental means by whichto control metabolic rate at the level of the intact animalis considered next. In the case of vertebrates we suggest thatthe organismic level of control might operate throughthe rateof capillary blood flow, as proposed in the Flow Theory of Coulson(1986): by controlling the rate at which the organized enzymearrays within the cells are perfused with substrate, cellularmetabolic rates could be set throughout the organism in an integratedfashion. Although there are problems with this linkage the interestingpossibility arises that the metabolic rate of individual cellsmay be subservient to the organism, being driven not so muchby the well known intracellular controls of concentration-based-biochemistryas by the flow of nutrients through the cells.     

Intracellular zinc during cell activation and zinc deficiency

IntroductionZinc is an essential trace element having manifold functions within living cells. Zinc deficiency but also zinc excess impairs cell-specific functions whereas a balanced zinc level is required for an adequate cell behavior.Material and methodsThis study deals with the impact of cellular priming due to stimulation with interleukin (IL)-1, IL-2, IL-4, IL-6 or the chemokine CXCL12a and its subsequent influence on the intracellular free zinc concentration. Since cellular priming and activation is essential for proper immunological reactions, and across that highly cell-type specific, we investigated T cells, B cells, and peripheral blood mononuclear cells (PBMCs). Additionally, alterations of the intracellular zinc content was investigated by inducing zinc deficiency using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) with subsequent re-supplementation of zinc, hence generating an intracellular zinc flux. Evaluation of zinc staining with FluoZin3-AM, Zinpyr-1 and Zinquin was done by flow cytometry or by fluorescence microscopy.ResultsOur results indicate that cellular priming for different periods of time (10 minutes/one hour) causes decreased intracellular free zinc concentrations in the FluoZin3-AM staining and increased zinc concentrations stained with Zinpyr-1. Furthermore, zinc supplementation after induced zinc deficiency leads to a fast and excessive rise of the intracellular free zinc levels in most cellular compartments.ConclusionOur study emphasizes the importance of zinc homeostasis and zinc distribution during cellular priming and for certain signaling cascades especially in T and B cells. Moreover, we demonstrated that zinc re-supplementation of zinc deficient cells results in significantly elevated intracellular free zinc concentrations compared to untreated controls. Hence, this underlines the need of a balanced zinc homeostasis for proper immune cell function.     

Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.     

Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

Summary The physiological consequences of overproduction of the homologous glycolytic enzyme 3-phosphoglycerate kinase (PGK), integrated in 80 PGK1 gene copies in the genome of Saccharomyces cerevisiae are described. This multiple integration and the strong PGK overproduction (maximum 47% of the total soluble cell protein) do not affect the maximal specific growth rate, but cause 40% reduction of the molar growth yield, compared with that of the wild-type host. The extra energy that is needed for protein overproduction is mainly provided by extra fermentation (respirofermentative growth), but respiration is also elevated compared with the reference strains. The increase in the specific oxygen uptake rate indicates that the respiratory capacity of the yeasts is higher than that in the wild-type host, in which the limited capacity of respiration is generally supposed to be at its maximal level at the critical dilution rate, and is thus responsible for the switch to respirofermentative growth. In a medium PGK1 gene copy integrant (about 25 copies), overproduction of 10%–12% PGK has a stimulating effect on the growth yield and energy efficiency. In these cells the growth benefits of overproduction of the glycolytic enzyme are higher than the disadvantages of extra protein synthesis. The overproduction of PGK has also consequences for the glucose affinity of the yeasts: In the more overproducing strain the K _s is increased, compared to its reference strains. Elimination of strong overproducing cells from a glucose-limited chemostat culture is caused by two factors: (a) the excision of the PGK genes from the genome, which is of minor importance for wash-out, but the induction process for this overall decline of overproduction, and (b) the physiological selection process for less overproducing cells, caused by differences in affinity for glucose, most obvious at µ 1/2µ_max. However in batch culture and in a chemostat at low specific growth rates, all the overproducing strains show high genetic stability and constantly provide high PGK quantities.Offprint requests to: P. C. van der Aar     

Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.     

The physiology of corticotropin-releasing hormone deficiency in mice

A review of the generation and characterization of corticotropin-releasing hormone (CRH)-deficient mice is presented. The studies summarized demonstrate the central role of CRH in the pituitary-adrenal axis response to stress, circadian stimulation, and glucocorticoid withdrawal. Additionally, pro-inflammatory actions of CRH at sites of local inflammation are given further support. In contrast, behavioral effects during stress that had been ascribed to CRH action are not altered in CRH-deficient mice. The normal behavioral response to stress in CRH-deficient mice strongly suggests the importance of other, possibly as yet undiscovered, CRH-like molecules.     

Consequences of hazardous dietary calcium deficiency for fattening bulls

Background  

Deficient mineral supplementation on a feedlot farm resulted in severe clinical manifestations in fattening bulls. Animals mistakenly received only 60–70% of the recommended calcium intake, while simultaneously receiving twice the amount of phosphorus recommended. Thus, the dietary Ca/P ratio was severely distorted. After approximately six months on such a diet, four fattening bulls were euthanized because of severe lameness and 15% of other animals on the farm were having clinical leg problems. Veterinary consultation revealed the mistake in mineral supplementation.     

Potassium magnesium homeostasis: physiology, pathophysiology, clinical consequences of deficiency and pharmacological correction

The metabolism of K and Mg is closely linked. Mg deficiency may arise together with and contribute to the persistence of K deficiency. Isolated disturbances of K balance do not produce secondary abnormalities in Mg homeostasis. In contrast, primary disturbances in Mg balance, particularly Mg depletion, produce secondary K depletion. This appears to result from an inability of the cell to maintain the normally high intracellular concentration of K, perhaps as a result of an increase in membrane permeability to K and / or inhibition of Na+-K+-ATPase. Cases of Mg deficiency accompanying with Mg-dependent or -independent K deficiency are not uncommon among the general population. K and Mg deficiencies are found in patients with chronic alcoholism, cardiac diseases, diabetes mellitus (type II), genetic forms of renal potassium and magnesium wasting (Gitelman's and Bartter's syndromes), severe diarrhea and vomiting, malnutrition, during therapy with some kind of drugs. Various K-Mg salts allowing simultaneously eliminating deficiency of Mg and K are described in the literature. K-Mg aspartate is most distributed among K-Mg salts. It can be used as adjuvant therapy in ischaemic heart disease (in angina pectoris and conditions after myocardial infarction), prophylaxis and adjuvant therapy of cardiac arrhythmia (e.g. prevention of toxic symptoms during therapy with digoxin). Differences in metabolism and utilisation of D- and L-amino acids probably may effect on pharmacological properties of K-Mg L- and D-aspartates, and what is more pharmacological doses of Mg and K salts may induce toxicity which differs according to the nature of the anions. In our research it was established, that L-aspartate salts are better delivery forms for cations such as Mg and K than D-aspartate salts. K-Mg L-aspartate can be more beneficial in the treatment of several forms of primary Mg and K deficiency than K-Mg DL-aspartate and K-Mg D-aspartate.     

Consequences of metamorphosis for the locomotor performance and thermal physiology of the newt Triturus cristatus

During metamorphosis, most amphibians undergo rapid shifts in their morphology that allow them to move from an aquatic to a more terrestrial existence. Two important challenges associated with this shift in habitat are the necessity to switch from an aquatic to terrestrial mode of locomotion and changes in the thermal environment. In this study, I investigated the consequences of metamorphosis to the burst swimming and running performance of the European newt Triturus cristatus to determine the nature and magnitude of any locomotor trade-offs that occur across life-history stages. In addition, I investigated whether there were any shifts in the thermal dependence of performance between life-history stages of T. cristatus to compensate for changes in their thermal environment during metamorphosis. A trade-off between swimming and running performance was detected across life-history stages, with metamorphosis resulting in a simultaneous decrease in swimming and increase in running performance. Although the terrestrial habitat of postmetamorphic stages of the newt T. cristatus experienced greater daily fluctuations in temperature than the aquatic habitat of the larval stage, no differences in thermal sensitivity of locomotor performance were detected between the larval aquatic and postmetamorphic stages. The absence of variation across life-history stages of T. cristatus may indicate that thermal sensitivity may be a conservative trait across ontogenetic stages in amphibians, but further studies are required to investigate this assertion.