Fetuin: an acute phase protein in cattle

Summary Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin — ₂HS glycoprotein — has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Bovis bovis) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30% in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle.Abbreviations ₂HS ₂HS glycoprotein - AP acute phase     

Steroid hormones as mediators of neural plasticity

Steroid and thyroid hormone receptors are expressed in the developing brain and persist throughout adult life. They mediate a variety of effects on the brain, ranging from developmental effects of thyroid hormone and the process of sexual differentiation to the cyclic changes during reproductive cycles in adult female animals. This review summarizes data from the author's laboratory on three topics: (1) actions of extradiol and progesterone on the ventromedial nucleus of the hypothalamus in adult female and male rats, showing both the cyclicity and the consequences of brain sexual differentiation; (2) actions of estradiol on the cholinergic neurons of the basal forebrain of the female and male rat, reflecting the plasticity of the adult cholinergic system as well as sex differences which are developmentally programmed; and (3) diverse actions of estrogens, thyroid hormone and glucocorticoids on the morphology of hippocampal neurons. The review concludes by discussing the interactions between "organizational" (i.e. developmental) effects and the "activational" effects of steroids on the mature nervous system in relation to the environmental control of brain gene expression.     

Cytochrome c4 from Pseudomonas aeruginosa

The dihaem cytochrome c₄ from Pseudomonas aeruginosa has been crystallized in space group P6₅22 with cell dimensions $\text{a = b = 62.4}\text{A}\text{?}\text{, c = 174.2}\text{A}\text{?}$, and one molecule per asymmetric unit. Two heavy-atom derivatives, UO₂(NO₃)₂ and K₂Pt(NO₂)₄, which substitute at one and three sites, respectively, have allowed a low-resolution electron density map to be obtained. This shows clearly the two domains of the molecule.     

Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. Thus immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.     

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.     

Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.     

Quantitative initiation of microtubule assembly by chromosomes from Chinese hamster ovary cells

The microtubule nucleating capacity of chromosomes was tested in vitro in lysates of Chinese hamster ovary cells. Colcemid-blocked mitotic cells were lysed with the detergent Triton X-100, incubated with exogenous porcine brain tubulin, attached to electron microscope grids and observed as whole-mounts. Under suitable conditions, greater than 98% of the chromosomes gave rise to microtubules at their kinetochore regions, thus unequivocally demonstrating that chromosomes are competent to initiate specifically microtubule formation. The average number of microtubules that polymerized onto a chromosome was 8 +/- 5, and greater than 36% of the chromosomes had between 10 and 19 microtubules per kinetochore region. We conclude that under the lysis conditions employed, virtually all the chromosomes retain their kinetochores, and that the kinetochores retain a substantial fraction of their microtubule nucleating capacity.