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.     

Effects of fenvalerate and azinphosmethyl on two-spotted spider mite and phytoseiid mites

The pyrethroid fenvalerate showed significantly faster activity against adult two-spotted spider mite Tetranychus urticae Koch c.f. azinphosmethyl using broad bean leaf discs sprayed in a Potter tower. LC₅₀s for fenvalerate were similar at 24 and 48 hr (0.056 and 0.051g AI/1) while LC₅₀s for azinphosmethyl were significantly different at 24 and 48 hr (0.72 and 0.38g AI/1, respectively). Mortality was partitioned to run-off and direct mortality. Fenvalerate showed an increasing contribution to mortality by run-off with increasing concentration. Increasing concentrations of azinphosmethyl had no effect on the proportion of T. urticae running off the discs. Fenvalerate inhibited egg production c.f. azinphosmethyl (60% and 20% inhibition respectively c.f. control after 24hr). The effect was not permanent. Carbaryl showed no acaricidal or inhibitory effects at 1g AI/1. T. urticae detected fenvalerate residues as reflected by choice of oviposition sites on untreated halves of leaf discs c.f. treated halves. Azinphosmethyl had no effect on oviposition preference. Phytoseiid mites were highly sensitive to fenvalerate residues. Predators moved off the test arena into sticky barriers after feeding on fenvalerate-treated eggs or walking on glass slides treated at 0.00005g AI/1.

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Résumé Les insecticides pyrethroïdes ont été utilisés pour lutter contre les pullulations d'araignées rouges. Cette note examine les réponses de Tetranychus urticae Koch et des prédateurs phytoseiidés résistants aux organo-phosphorés, Amblyseius fallacis Garman et Typhlodromus occidentalis Nesbitt, au fenvalerate (pyrethroïde) et à l'azinphosmethyl (organophosphate). Quelques essais avec du carbaryl sont indiqués.Une femelle adulte de T. urticae est placée sur une rondelle de feuille de Phaseolus vulgaris L. pulvérisée dans une tour de Potter.Les résultats sur la mortalité en fonction de la dose obtenus montrent une activité plus rapide du fenvalerate que de l'azinphosmethyl. Les DL₅₀ du fenvalerate (0,056 et 0,051g AI/I) sont les mêmes à 24 et 48 h, tandis que l'azinphosmethyl montre une activité plus lente (DL₅₀ de 0,72 et 0,38g AI/I à 24 et 48 h). La mortalité se partage entre la sortie de la rondelle et la mortalité in situ.Le fenvalerate provoque une plus forte répulsion que l'azinphosmethyl. Contrairement à l'azinphosmethyl le fenvalerate inhibe la production d'oeufs 60% et 20% d'inhibition à DL50 au bout de 24 h par rapport au témoin. Cette inhibition n'est pas permanente. Le carbaryl n'a pas d'effets inhibiteur ou acaricide à 1g AI/l.Les femelles adultes de T. urticae décèlent les résidus de fenvalerate sur les rondelles et pondent leurs oeufs sur les moitiés non traitées ou traitées à l'azinphosmethyl.Les Phytoseiides sont très sensibles aux résidus de fenvalerate. Après consommation d'oeufs traités, A. fallacis est incapable d'éviter des bandes gluantes. T. occidentalis décèle des traitements à 0,00005g AI/l en quittant les lames traitées par les bandes gluantes.

     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.

Studies reported in the preceding paper (Trowbridge and Hyman, 1979) have demonstrated that Thy-1^? mutant lymphoma cells of the class E complementation group lack the normal high molecular weight lipid-linked oligosaccharide, but instead accumulate two smaller species termed I and II. This paper reports studies which elucidate the structures of lipid-linked oligosaccharides I and II. By subjecting oligosaccharides radiolabeled with ³H-mannose, ³H-glucose or ³H-glucosamine to methylation, acetolysis, periodate oxidation and exoglycosidase digestion, the structures were shown to be: where R = GlcNac B1,4(3) GlcNAc. A comparison of I and II with lipid-linked oligosaccharides from normal Chinese hamster ovary cells indicates that both I and II are normal biosynthetic intermediates. On the basis of these data we suggest that the defect in the class E mutant cells is the lack of an α1,3 mannosyltransferase involved in the conversion of the Man₅GlcNAc₂ lipid-linked oligosaccharide to the Man₆GlcNAc₂ intermediate. It is also impossible that the same enzyme is involved in conversion of the Glc₃Man₅GlcNAc₂ lipid-linked oligosaccharide to Glc₃Man₆GlcNAc₂. The latter reaction, however, has not yet been demonstrated in normal cells.     

Maternal X chromosome expression in mouse chorionic ectoderm

An electrophoretic variant of the X-linked enzyme phosphoglycerate kinase (PGK-1) has been used to study regulation of X chromosome expression in the diploid derivatives of the trophectoderm at 8–8.5 days post coitum in the mouse. These derivatives included the chorionic ectoderm and the polar trophoblast. The biochemical analysis suggests that only the maternally derived X chromosome (X^m) is expressed in the diploid trophectoderm derivatives. Cell selection and maternal tissue contamination were ruled out as possible causes of the observed X^m expression. From these and other results, we conclude that all derivatives of the trophectoderm, along with the primitive endoderm, express only X^m, whereas derivatives of the primitive ectoderm show random X chromosome expression.     

Feeding,energy flow and soil turnover in the desert isopod,Hemilepistus reaumuri

The desert woodlouse Hemilepistus reaumuri is one of the most abundant macroscopic invertebrates in North Africa, the Arabian steppes, semi-desert and desert, and the Negev desert Israel.The main purposes of our study were:     

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.