3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Mathematical Model of Leukemic-Cell Circulation in the Mouse

L1210 leukemic cells injected in vivo are eliminated from the blood and disintegrated in organs such as the lungs and liver. We present a compartmental model which reproduces one type of in vivo experiment, based on the so-called perfusion curves. Although the data are not complete and some are only approximated, modeling gives a consistent picture of the process.     

Stand microclimate and physiological activity of tree leaves in an oak-hornbeam forest

Summary In an uneven-aged, multi-species oak-hornbeam forest at Báb, SW Slovakia (former IBP Forest Research Site), a series of micrometeorological and ecophysiological measurements started in 1985. The aims of the work are to improve understanding of physiological processes (photosynthesis, respiration, and transpiration) of adult trees and stand microclimate, to collect data for simulation of the canopy (stand) photosynthesis and for ecological synthesis of the functioning of the forest ecosystem. In this paper, photosynthetically active radiation (PAR), air temperature (AT) and relative humidity (RH), wind speed (WS), and CO₂ concentration ([CO₂]) in and above the forest are characterized for the fully leaved season, using diurnal courses, vertical profiles and isodiagrams (isopleths). Approximately 50% of incident PAR was absorbed by the upper 4–5 m layer of leaves and only approximately 5% or less penetrated to the forest floor. Vertical gradients of AT and RH were generally low, but large differences in diurnal ranges of AT and RH were observed between vertical levels. The upper leaf canopy greatly reduced WS, and at a height of about 14 m above the ground it was close to zero. The highest diurnal [CO₂] maximum and variations occurred at 1 m above the ground, and the lowest above the forest. In good light conditions in the forest, the entire leaf canopy (overstorey and understorey canopy) is a large sink of CO₂. At night the forest stand is a source of CO₂, the largest internal source being the soil and forest floor.     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

Inhibitors and activators of ADP-ribosylation reactions

ADP-ribosylation reaction, that is the transfer of the ADP-ribose moiety of NAD⁺ to acceptor protein, is catalyzed by two classes of ADP-ribosyltransferases,i.e., poly(ADP-ribose) synthetase and mono (ADP-ribosyl)transferases. These two types differ not only in the number of transferring ADP-ribose units but also in the acceptor amino acid(s) and protein. Their in hibitors, particularly those of poly(ADP-ribose) synthetase, have been successfully employed in studies on biological functions of the enzymes and other related fields of research. Recently, we found many potent and specific inhibitors of poly-(ADP-ribose) synthetase, and broadened their chemical as well as biochemical variety. More recently, we found several potent inhibitors of arginine-specific mono(ADP-ribosyl)transferases and activators of poly(ADP-ribose) synthetase.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Biomass yields of Chlorella from iron (Y x/Fe) in iron-limited batch cultures

The maximum biomass in iron-limited photosynthetic batch cultures of chlorella increased as the logarithm of the iron concentration. The growth yield from iron (Y _x/Fe) showed a marked inverse relation to the specific growth rate. The maximum biomass yield, g dry biomass/g iron consumed, was 7.5x10³ with specific growth rate 0.108 h^-1; the minimum was 0.79×10³ with specific growth rate 0.145 h^-1. The maximum specific growth rate in the exponential phase of Fe limited cultures varied as the initial Fe concentration. Fe-limited growth made the cells adhere to a glass surface.Abbreviation O.D. optical density     

The effect of experimental hyperthyroidism on renal and adrenal weight increase in mice.

The mouse kidneys are enlarged after the administration of thyroxine and this influence is not mediated through androgens. The administration of thyroxine increased the weight of the adrenals and the level of plasma corticosterone. Besides the direct effect of the thyroid hormones on the kidney, our findings indicate that the excess of triiodothyronine and thyroxine stimulates the activity of adrenals indirectly and evokes hyperadrenocorticism which could be related to the action of adrenal steroids on kidney function and kidney growth. In accordance with the above mentioned hypothesis it has been shown that aminoglutethimide, a potent blocker of adrenal steroidogenesis, decreases the level of plasma corticosterone and inhibits the enlargement of the kidney in hyperthyroid mice in spite of the high serum thyroxine values.     

Comparison of different methods of glucose oxidase immobilization

Glucose oxidase was immobilized by covalent bond to two basic types of sorbents—glycidylmethacrylate copolymers and bead cellulose. These two types of carries were chemically modified, if needed, by the employing various procedures and subsequently used in the immobilization of native and oxidized glucose oxidase. The samples thus obtained were compared with those of immobilized glucose oxidase bound onto some common carriers. Samples which possessed not only a high absolute activity but also adequate mechanical and flow properties were characterized in greater detail with respect to the immobilization efficiency and kinetic properties of bound glucose oxidase.