The physical mechanism of calcium pump regulation in the heart.

The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban increases the rotational mobility of the Ca-ATPase in the sarcoplasmic reticulum bilayer, due to a decrease in large-scale protein association, with a [Ca2+] dependence parallel to that of enzyme activation. These results support a model in which phospholamban phosphorylation or calcium free the enzyme from a kinetically unfavorable associated state.     

Hormone Effects on the Membrane Potential and on Sucrose-Induced Depolarization of Young Citrus Leaves

The effect of IAA, GA₃ and ABA on transmembrane potential difference(Em) and on sucrose-induced depolarization has been studiedin young Citrus leaves. The addition of any of these hormonesto the perfusion solution (short-term experiments) did not affectEm or sucrose-induced depolarization. Hormonal treatments ofyoung leaves on the tree resulted, after 4 to 16 days (long-termexperiments), in an increase of Em for GA₃- and ABA-treatedleaves, while in IAA-treated ones no hyperpolarization was found.Only in ABA treated leaves this membrane hyperpolarization couldbe related to an enhancement of sucrose uptake. (Received April 28, 1992; Accepted September 21, 1992)     

A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.

A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.     

Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes

Summary The synthetic peptides somatostatin (SRIF) and growth hormone-releasing hormone (GRH) were coupled directly to colloidal gold of different particle sizes. Both conjugates were biologically active in displacing the corresponding radiolabeled hormones from high affinity binding sites in pituitary membranes. Release of growth hormone (GH) from cultured anterior pituitary cells was modulated by both conjugates alone or in combination. Ultrastructural studies were performed with cells incubated at 4° C (2 h) and 37° C (2 min-2 h) with one of the labeled peptides or their combination. Somatotropes were identified by immunostaining with anti-rGH followed by protein A-ferritin, thus obtaining a triple labeling. Both hormone conjugates were internalized in different vesicles in the beginning but accumulated during longer incubation times in the same compartment. The secretory vesicles and the nucleus were not labeled by any hormone conjugate. In contrast to SRIF-gold, the uptake of GRH-gold conjugate decreased with longer incubation times. This effect could be neutralized by simulatenous incubation of the somatotropes with both regulating hormones. Hence, whereas the binding and internalization of SRIF by somatotropes do not seem to be influenced by GRH, the corresponding processes for GRH are stimulated by the presence of SRIF.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.