Syntheses and properties of circular dichroism active phospholipids

A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of approximately 550 A diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phospholipid having two trans-azobenzene chromophores showed an extremely large CD enhancement even well above Tc. This CD enhancement was drastically changed by the presence of cis-azobenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.     

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical evidence of progestin target cells in the pituitary gland of ovariectomized rat

Progestin (P) target cells were identified in the pituitary gland of gonadectomized female rats which had been primed with estrogen (E). P staining was localized using the immunohistochemical avidin-biotin-peroxidase (ABP) complex method. Dark brown precipitates were primarily found over the cytoplasm of cells in the pars distalis, but not in the pars intermedia nor in the pars nervosa. The majority of P-sensitive cells in the pars distalis were identical with luteotrophs, a few being lactotrophs. These observations suggest a role of P in the regulation of production and secretion of gonadotrophins in the pituitary glands of female rats.     

Heterogeneity of adrenocortical ferredoxin

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.     

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. I. the Modes of Diploidization in the Gynogenesis Induced by a Male-Sterile Mutant, ms(3)K81

Sperm that are produced by males homozygous for ms(3)K81 , a male sterile mutant of Drosophila melanogaster, are defective in syngamy but are capable of activating eggs to develop gynogenetically. The activated eggs usually produce haploid embryos, but a small fraction (10 ^-4–10^-5) of them give rise to diploid impaternate adults. To know the cytological mechanisms by which these impaternates restore diploidy, the genotypes of impaternate progeny obtained from females doubly heterozygous for visible markers were examined. The results show that, as generally found among parthenogenetic Drosophila, diploidy is restored after completing meiosis either by pronuclear fusion or by gamete duplication (doubling of a haploid cleavage nucleus). The fusion of two nonsister nuclei following meiosis II (central fusion) was indicated to be a predominant mode of diploidization in this species. Two meiotic mutants, mei-9 and mei-S332, which are known to greatly increase meiotic nondisjunction, did not cause an increased incidence of impaternates. This seems to exclude the possibility that some impaternates might have been derived from diploid egg nuclei produced through nondisjunction.     

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.     

Changes in plasma fibronectin levels in thyroid diseases

The plasma levels of fibronectin (Fn) have been measured in normal subjects and in patients with thyroid diseases. The mean plasma Fn levels in 62 normal adults was 32.0 +/- 6.0 mg/dl, whereas it was elevated to 62.6 +/- 16.1 mg/dl (mean +/- SD) in 25 patients with hyperthyroidism and decreased to 19.2 +/- 8.0 mg/dl in 9 patients with hypothyroidism. The 9 patients with simple goiter have normal values of 29.1 +/- 8.0 mg/dl. With the administration of anti-thyroid drugs, plasma Fn levels normalized, with a time lag, in parallel with normalization of the thyroid function. Positive correlation was obtained between Fn levels and serum levels of triiodothyronine (T3) and thyroxine (T4). The present findings indicate that measurement of plasma Fn both in the basal state and during treatment provides evidence of altered Fn metabolism in thyroid diseases and serves to follow up the effect of treatment.