Modulation of phosphatidylcholine synthesis in vitro. Inhibition of diacylglycerol cholinephosphotransferase and lysophosphatidylcholine acyltransferase by centrophenoxine and neophenoxine

1,2-Diacyl-sn-glycerol : CDPcholine cholinephosphotransferase (EC 2.7.8.2) and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) activities of rat liver microsomes can be inhibited by centrophenoxine (N,N-dimethylaminoethyl p-chlorophenoxyacetate). This inhibition is brought about by the intact centrophenoxine molecule rather than by the products of hydrolysis. A nonhydrolyzable ether analog of centrophenoxine was synthesized (neophenoxine; N,N-dimethylaminoethyl p-chlorophenoxyethyl ether) and proved most effective in inhibiting the two routes of phosphatidylcholine biosynthesis. While 50% inhibition of the cholinephosphotransferase was attained at 5 mM neophenoxine, 50% inhibition of the acyltransferase required 0.6 mM neophenoxine levels only. Inhibition of the cholinephosphotransferase (Ki approximately 1.5 mM) and the acyltransferase (Ki approximately 1 mM) by neophenoxine was shown to be noncompetitive. Other membrane-bound enzymes, such as glucose-6-phosphatase, monoacylglycerol lipase, alkaline phosphatase or phospholipase A2 were not affected by the inhibitors. Because of this specificity, and because of the high affinity of the microsomal membrane for such agents, centrophenoxine and neophenoxine should prove useful for controlling phosphatidylcholine synthesis and for modulating the phosphatidylcholine deacylation-reacylation cycle.     

The specificity of purified porcine pancreatic elastase

An electrophoretically homogeneous elastase preparation free from tryptic and chymotryptic activities was obtained by chromatography on DEAE-Sephadex and CM-cellulose. This preparation exhibits a narrower specificity towards peptide bonds than that observed by Naughton & Sanger (1961). With oxidized insulin B chain as substrate, the fastest breaks occur between alanine-14 and leucine-15 and between valine-18 and cysteic acid-19. The bond between glycine-23 and phenylalanine-24 is also efficiently hydrolysed. Other bonds hydrolysed are that between valine-12 and glutamic acid-13 and that between serine-9 and histidine-10. Oxidized insulin A chain is hydrolysed only at one of two points, between alanine-8 and serine-9 or between serine-12 and leucine-13, and the rate of hydrolysis is very low.     

OBSERVATIONS ON METAPHLOEM IN THE VEGETATIVE PARTS OF PALMS

Metaphloem was studied in available vegetative parts of 374 species in 164 genera of palms. Sieve elements usually have compound sieve plates except in the subfamilies Lepidocaryoideae and Nypoideae. Sieve elements in roots usually have oblique to very oblique end walls, whereas in stems and leaves they have transverse to oblique walls. Within a phloem strand the degree of compounding of a sieve plate is directly correlated with element diameter. Plastids are normally present in functioning, enucleate sieve elements. Small quantities of “slime” substances have been detected in young sieve elements in stems and petioles of a few species. Many sieve plates in functioning sieve elements lacked callose in materials quick-killed in liquid nitrogen or chilled acetic-alcohol. Definitive callose is confined to sieve elements just before their obliteration. Sieve tubes in leaf and stem are usually ensheathed by contiguous parenchyma cells while those in root have very few contiguous parenchyma cells. Two types of contiguous parenchyma cells can be distinguished by difference in cytoplasmic density, especially with the electron microscope. Cells with denser cytoplasm are interpreted as companion cells. Lignified contiguous parenchyma cells are occasionally present in metaphloem of petioles. The possible diagnostic and taxonomic features of metaphloem are discussed.     

Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae).

Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.     

Direct modification of low density lipoprotein by the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid.

We have previously reported that the spin trap alpha-phenyl-tert-butyl nitrone (PBN) inhibited the oxidative modification of low density lipoprotein (LDL) (Kalyanaraman, B., Antholine, W.E. and Parthasarathy, S. (1990) Biochim. Biophys. Acta 1035, 286-292). In the present study, we report that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS), a water-soluble spin trap, also inhibited the oxidation of LDL as measured by the formation of thiobarbituric acid reactive substances (TBARS). However, when compared with LDL incubated without DBNBS, the DBNBS-incubated LDL showed increased negative charge on agarose gel electrophoresis and was avidly degraded by mouse peritoneal macrophages. Despite the suggestion of biological modification, there was no decrease in lysine-amino groups in DBNBS-incubated LDL. Furthermore, reductively methylated LDL in which more than 85% of the amino group of lysines was blocked, was also modified by DBNBS. A sulfonic acid analog of PBN failed to modify LDL in a similar manner, suggesting that the presence of sulfonic acid alone does not ensure modification. When LDL was incubated with DBNBS, radical adducts associated with both lipid and protein were detected by electron paramagnetic resonance (EPR) technique. It is suggested that DBNBS may bind to the apoprotein B100 and lipids of LDL by a lysine-independent mechanism resulting in increased recognition and degradation by macrophages. The present work offers a novel approach for rapid modification of LDL.     

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

Summary High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.     

Oxidation of low-density lipoprotein by thiol compounds leads to its recognition by the acetyl LDL receptor

Reduced glutathione and other compounds with free -SH groups promoted the oxidation of low-density lipoprotein (LDL) in the absence of cells in Ham's F-10 medium. In contrast, compounds in which the thiol groups were oxidized or blocked were ineffective in oxidizing LDL. Thiol-induced modification of LDL did not occur in media lacking in redox metals. It is suggested that thiols react with redox metal, generating thiol- and oxygen-derived free radicals that promote modification of LDL.     

A circular dichroism study of (+) gossypol binding to proteins

The circular dichroism bands of (+) gossypol in the spectral region 300-400 nm have been shown to be sensitive to interactions with proteins. Using CD spectroscopy, gossypol has been shown to interact with lactate dehydrogenase, malate dehydrogenase, alkaline phosphatase, lysozyme, protamine and poly-L-lysine. Binding to proteins generally results in a pronounced red shift of the long wavelength CD band (approximately 380-430 nm) accompanied by a reduction in ellipticity. The changes in spectral parameters of the 1Lb binaphthyl transition may reflect a distortion from a nearly perpendicular gossypol conformation, on binding to proteins.