Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concomitant changes in intramembrane particle size during photoinhibition of Chlamydomonas reinhardtii

Light-dependent reduction of the plastoquinone pool regulates the activity of the thylakoid-bound protein kinase which phosphorylates the light harvesting chlorophyll a,b-protein complex (LHC II) and regulates energy distribution between photosystems II (PS II) and I (Staehelin, L. A., and C. J. Arntzen, 1983, J. Cell Biol., 97:1327-1337). Since reduction of plastoquinone by PS II is abolished in photoinhibited thylakoids due to loss of the secondary electron acceptor QB protein (Kyle, D. J., I. Ohad, and C. J. Arntzen, 1984, Proc. Natl. Acad. Sci. USA, 81:4070-4074), it was of interest to examine the activity of the LHC II protein kinase system during photoinhibition and recovery of PS II activity. The kinase activity was assessed both in vivo and in vitro in Chlamydomonas cells exposed to high light intensity (photoinhibition) and recovery at low light intensity. The kinase activity was progressively reduced during photoinhibition and became undetectable after 90 min. The inactive LHC II-kinase system could not be reactivated in vitro either by light or by reduction of the plastoquinone pool following addition of reduced duroquinone (TMQH2). The LHC II polypeptides were dephosphorylated in vivo when cells, prelabeled with [32P]orthophosphate before exposure to high light intensity, were transferred to photoinhibiting light in the presence of [32P]orthophosphate. In vivo recovery of the LHC II-kinase activity, elicited by the addition of TMQH2 to the assay system, did not require restoration of QB-dependent electron flow or de novo protein synthesis, either in the cytoplasm or in the chloroplast. Mild sonication of thylakoids isolated from photoinhibited cells restored the ability of the LHC II protein kinase system to be activated in vitro by addition to TMQH2. Restoration of the light-activated LHC-II kinase required recovery of QB-dependent electron flow. At the structural level, photoinhibition did not affect the ratio of grana/stroma thylakoids. A reduction of approximately 20% of the 11-17-nm intramembrane particles and an equivalent increase in the number of 6-10.5-nm particles was observed on the E-fracture faces of stacked thylakoid membranes. Similar but smaller changes were observed also on the E-fracture faces of unstacked thylakoid membranes (more 10-14-nm and less 6-9-nm particles) and P-fracture faces of stacked thylakoid membranes (more 6-8- and less 9.5-13-nm particles). All these structural changes were reversed to normal values during recovery of PS II activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

Synthesis of gamma-monodentate and beta, gamma-bidentate CrITP. Isolation and characterization of the two chelation isomers and the individual diastereomers of beta, gamma-bidentate CrITP

The two chelation isomers of CrITP, gamma-monodentate and beta, gamma-bidentate CrITP, as well as the diastereomers of beta, gamma-bidentate CrITP were synthesized, isolated, and characterized. Synthesis of these complexes was done using pH titration methods similar to that described by Cleland [W.W. Cleland, Methods Enzymol. 87, 159 (1982)], and separation of the two chelation isomers was accomplished with DEAE-sephadex A-25 using 0-0.3 N linear HCl gradient. Diastereomer separation (analytical and preparative scales) of beta, gamma-bidentate CrITP using reverse-phase high-performance liquid chromatography, and then analysis of the diastereomers with circular dichroism spectroscopy, shows four diastereomers that exist as two pairs of mirror-image isomers, similar to the four diastereomers of beta, gamma-bidentate CrATP as presented by Dunaway-Mariano and Cleland [D. Dunaway-Mariano and W.W. Cleland, Biochemistry 19, 1496 (1980)]. Reverse-phase high-performance liquid chromatography analysis of gamma-monodentate CrITP shows the presence of two major peaks, both of which convert to beta, gamma-bidentate CrITP upon incubation at pH 6.0 for 1 hr.     

Systematic investigation of degradation effects on spin-lattice relaxation times in mouse-liver by low resolution NMR

In spite of numerous work on in vitro proton relaxation time investigations of biological tissue, many questions still remain open. In this study we focused on spin-lattice (T1) relaxation time measurements of mouse liver tissue in order to estimate the time-after-excision effects. The post mortem behaviour of excised tissue was measured up to four hours in intervals of about nine minutes. The time course of liver T1 was determined for different temperatures (4 degrees-40 degrees C) for female mice and the effect of starvation (up to 48 hours) on the time course of T1 was investigated for male and female mice at 37 degrees C. We obtained significant differences in liver T1 time course after excision due to different physiological states like sex, starvation and circadian rhythm.     

The NADH-dehydrogenase subunit 5 gene in Oenothera mitochondria contains two introns and is co-transcribed with the 5 S rRNA gene

Summary The gene encoding subunit 5 of the NADH dehydrogenase complex (ND 5) has been identified in Oenothera mitochondria from a cDNA clone. The coding region is interrupted by a type II intron of 850 nucleotides and a second intervening sequence of 357 nucleotides. Genomic sequence rearrangement within the first intron creates a nontranscribed partial copy of the gene. The intact ND 5 gene is transcribed in a complex pattern with mRNAs including the 5 S rRNA sequence. Excision of the two introns appears to proceed slowly in vivo since the steady state mitochondrial RNA contains significant proportions of unprocessed precursor molecules.     

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.