Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract The plasma membrane of Saccharomyces cerevisiae and Schizosaccharomyces pombe in stationary phase had abundant invaginations. A round uninvaginated area emerged before budding when S. cerevisiae cells were given fresh medium. Middle-sized buds had some invaginations, whereas the neck between the bud and mother had very few. S. pombe which has neither the neck nor the predetermined position to divide had no uninvaginated ring area even in long cells during elongation in fresh medium. However, an uninvaginated ring area emerged as the earliest noticeable stage of cytokinesis. The uninvaginated state of the plasma membrane appeared to be correlated with budding and cell division.     

Staining of living bacteria with rhodamine 123

Abstract It is possible to stain live bacteria with rhodamine 123 (R123). The stained fluorescent cells still keep the ability to replicate ( Staphylococcus aureus, Bordetella pertussis ) and to swim (e.g., Salmonella minnesota ). Dead cells or cells with a dissipated transmembrane potential showed markedly diminished fluorescence. Gram-negative strains were stained with different efficiency, presumably reflecting the different constitutions of the outer membrane.     

Molecular genetic and phenotypic alteration of Escherichia coli in natural water microcosms containing toxic chemicals

Abstract Microcosms of sterile Chesapeake Bay water were used to study effects of sub-lethal concentrations (1 μl/l) of nitrobenzene, m -cresol, and dibutyl phthalate on Escherichia coli H10407. E. coli remained viable during the 19-day test period in estuarine water, both in the presence and absence of the chemicals, long after it became non-culturable. Analysis of membrane proteins revealed changes in the protein composition. Carbohydrate and amino acid utilization was affected by these changes. Plasmids in E. coli H10407 could not be detected following microcosm exposure. When the cells were transferred to rich medium without toxic chemicals, growth resumed and plasmid bands were again detectable.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Kinetic analyses of the membrane-bound alkaline phosphatase activity of hymenolepis diminuta (Cestoda: Cyclophyllidea) in relation to development of the tapeworm in the definitive host

The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5'-nucleotidase activities associated with the brush-border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6-, 12-, and 18-d-old H diminuta, and individual pieces of 18-d-old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows.     

B-cells of the synovial membrane. II. Differentiation during development of the synovial cavity in the mouse

Summary Study of pre- and postnatal development of the metatarsophalangeal joint of the mouse shows that the synovial cavity (SC) forms before any differentiation of the synovial mesenchyme. The primitive cleft results from degradation of a thin vascular mesenchymal layer in direct contact with the chondrogenic layers. Differentiation of the synovial membrane coincides with clarification of the SC (3rd to 6th day of postnatal life). When dilatation of the SC occurs (6th to 8th day), the two intimal cells types (A- and B-cells) are well identified. The B-cells already show typical features at day 6; their content of typical dense secretory vesicles is comparable to that of the adult B-cells at day 13. The specific secretory function of B-cells could be correlated with the particular structure of the intimal interstitial tissue and could account for the origin of some protein(s) of the synovial fluid.ERA 178 (Neuroendocrinologie Comparée) du CNRS et INSERM     

Hydroxycinnamic acid amides in fertile and cytoplasmic male sterile lines of maize

Hydroxycinnamic acid (HCA) amides in fertile and cytoplasmic male sterile lines of maize were determined in reproductive organs, developing grains and cobs. HCA amides occurred in large amounts in the anthers of fertile plants (line F7N) and were absent from the anthers of cytoplasmic male sterile lines (lines F7T and F7C). Restoration of fertility was associated with the production of these compounds (line FC31). Considerable variations were observed in the concentrations of HCA amides at different stages of growth and grain maturation. Changes of HCA amides in the grains which were to produce sterile plants followed a pattern similar to that obtained with the grains which were to produce fertile plants. Accumulation of HCA amides was substantially higher in fertile lines whatever their genotype (F7N, FC31 and F7T x FC31) than in sterile lines. Marked changes occurred in the HCA amide content of embryo and endosperm during grain development. Many changes in HCA amides were observed in cobs during development and maturation, but no substantial differences could be observed between fertile and sterile lines.     

Electron spin resonance study of the isolated lipid components from Blastocladiella emersonii zoospores

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.