Phosphorylation of the calcium ion-regulated thin filaments from vascular smooth muscle. A new regulatory mechanism?

The thin filaments of vascular smooth muscle (pig aorta) contain a Ca2+-sensitive regulatory system that resembles troponin-tropomyosin [Marston, Trevett & Walters (1980) Biochem. J. 185, 355-365]. Our thin-filament preparations also contain enzymes that phosphorylate and dephosphorylate a specific protein. Initial rate of phosphorylation was 0.42 +/- 0.10 (95% confidence limits) mumol of Pi/min per g of thin filaments; half-maximal incorporation was obtained in 4 1/2 min, and a maximum of 1.8 +/- 0.1 mumol of Pi/g of thin filaments was incorporated after 40 min (conditions: 1 mM-MgATP, 60 mM-MgATP, 60 mM-KCl, 10 mM-imidazole, pH 7.0, 5 mM-MgCl2, 10 mM-NaN3, 0.5 mM-dithiothreitol, 0.1 mM-CaCl2, 25 degrees C). On gel electrophoresis in polyacrylamide (4-30% gradient)/0.25% sodium dodecyl sulphate gel over 75% of protein-bound phosphate was in a single protein of mol.wt. 21000. On electrophoresis in polyacrylamide (8%)/6 M-urea (pH 8.6) gel the phosphoprotein remained at the origin. Phosphorylation was associated with an increase in the concentration of high-affinity (K congruent to 10(6) M-1) Ca2+-binding sites from 0.8-1.5 to 6.3 mumol of Ca2+/g of thin filaments. Phosphorylation also changed the regulatory properties of the skeletal-muscle myosin-aorta thin-filament MgATPase; maximum activity was unaltered, but the phosphorylated thin filaments required only 0.36 microM-Ca2+ for half-activation compared with 2.7 microM-Ca2+ for unphosphorylated thin filaments. The possible regulatory role of thin-filament phosphorylation is discussed.     

Neural cells in culture: models for the purification and the study of the effects of growth factors

Conclusion and Perspectives We have summarized several methodologies currently available for the detection and analysisof protein factors affecting neural cells. As more knowledge is gained concerning growthfactors and their influences on cells and as the demand for bioassays increases, methodologicalimprovements are likely to be aimed at 1) decreasing yet the assay time; 2) increasingsensitivity of the measurements of cell responses; 3) increasing the number of simultaneouscellular responses measurable. A new very promising technique involves the use of severalfluorochromes having different cellular localizations and the continuous measurement of theirevolution in living cells by fluorospectrophotometry.The cell-blot method can be expected to extend to fractions submitted to other forms ofchromatography (native electrophoresis, isoelectric focusing, agarose). Other modificationsmay allow the detection of antagonistic factors, by including a stimulatory factor in the blotculture medium and measuring the suppression of a cellular response by the blotted opposingactivity.     

Neurite-Promoting Factor in Conditioned Medium from RN22 Schwannoma Cultures: Bioassay, Fractionation, and Properties

Abstract: On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary Neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent.     

Molluscicidal saponins from Phytolacca icosandra

Five monodesmosidic serjanic acid saponins and a monodesmosidic spergulagenic acid saponin were isolated from an aqueous extract of the berries of Phytolacca icosandra. A methanol extract of the berries furnished three bidesmosidic serjanic acid glycosides. Their structures were established by spectroscopic (ES-MS, 1H NMR, COSY, HSQC, HMBC, 13C NMR) and chemical methods. The molluscicidal, spermicidal and haemolytic properties of the saponins were investigated.     

Antifungal and larvicidal cordiaquinones from the roots of Cordia curassavica

In addition to the known cordiaquinones A and B, two novel meroterpenoid naphthoquinones, named cordiaquinones J and K, have been isolated from the roots of Cordia curassavica. Their structures were elucidated by spectrometric methods including EI, D/CI mass spectrometry, 1H, 13C and 2D-NMR experiments. The four naphthoquinones demonstrated antifungal activities against Cladosporium cucumerinum, Candida albicans and toxic properties against larvae of the yellow fever-transmitting mosquito Aedes aegypti.     

On-line identification of unstable iridoids from Jamesbrittenia fodina by HPLC-MS and HPLC-NMR

HPLC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina (Wild) O. M. Hilliard (Scrophulariaceae) revealed the presence of different iridoid cinnamic esters; however, isolation of these constituents was prevented by instability problems. HPLC-UV-MS and HPLC-NMR analysis of the mixtures obtained after a tentative isolation indicated that, in the first instance, instability was due to a light-induced cis/trans isomerisation of the cinnamic moieties. Further investigation of related compounds showed an additional instability problem linked to other chemical transformations. A detailed HPLC-NMR-MS study of these fractions demonstrated that the modifications occurred on the rhamnose moiety of these iridoids. It could be concluded that the second type of instability was attributable to transesterification of the cinnamic moiety on the rhamnose unit. The recording of stop-flow HPLC-NMR spectra for specific HPLC peaks permitted the direct monitoring of these transformations. Based on these on-line data, six new unstable aucubin derivatives were efficiently characterised.     

What happens to shelter dogs? An analysis of data for 1 year from three Australian shelters

Annually, welfare shelters admit many dogs, including those whose caregivers surrender them or dogs who are strays. This article analyzes admission data from 3 metropolitan Australian shelters. The study collected data for a 1-year period and analyzed them to identify the characteristics of the typical shelter dog; patterns of relinquishment, sales, reclamation and euthanasia; and duration of stay and reasons underlying euthanasia, relinquishment, and postadoptive return. The study tracked more than 20,000 admissions during this period. To facilitate reclamation, the local Code of Practice requires a mandatory holding period for stray dogs; assessment for suitability for rehoming then occurs. Dogs failing the assessment are euthanized. Surrendered dogs can be assessed immediately. The Code of Practice also recommends that unsold dogs be euthanized 28 days postassessment. Typically, shelter dogs in Melbourne are strays, sexually entire, adult, small, and-usually-male. The majority of admissions are reclaimed or sold. Most reclamations occur within 4 days, and postadoptive return rates are low. That current desexing messages do not appear to have reached the owners of stray dogs to the same extent as they have other dog owners is a major finding, suggesting that a targeted education campaign may be required.     

Meiosis: cell-cycle controls shuffle and deal

Meiosis is the type of cell division that gives rise to eggs and sperm. Errors in the execution of this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Here, we review recent findings on how cell-cycle controls ensure the coordination of meiotic events, with a particular focus on the segregation of chromosomes.     

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.