A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.     

Effects of genetic and environmental host plant variation on the susceptibility of two noctuids toBacillus thuringiensis

TwoApium graveolens var.rapaceum (L.) cultivars that differ in their suitability for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) were used to examine the effect of genetic and seasonal environmental variation in host plant suitability on the efficacy ofBacillus thuringiensis subsp.kurstaki (Berliner). The effects of host plant genotype andB. thuringiensis were generally independent, so thatB. thuringiensis efficacy was greatest on the resistant host plant cultivar. Host plant suitability varied within growing season for both insect species but, while host plant suitability decreased with increasing plant age forT. ni, the response ofS. exigua to plant age was not as clear. Within season variation in host plant suitability affectedB. thuringiensis efficacy and the interaction betweenB. thuringiensis and host plant cultivar forS. exigua but not forT. ni. Soluble protein and Folin-Denis phenolic concentrations of host plant tissue were not correlated with changes in host plant suitability to either insect species.     

Bicyclic carbo- and heterocycles from an allylidene complex and cyclic 1,3-dienes by tandem cyclopropanation/cope rearrangement

The allylidene complex (CO)₅W=CH---C(Ph)=C(Ph)H (4) reacts with cyclopentadiene by stereospecific transfer of the carbene ligand to one of the two double bonds of cyclopentadiene to give a cis-divinylcyclopropane complex 5. The divinylcyclopropane ligand coordinates to the metal via the unsubstituted double bond. Addition of bromide to solutions of 5 gives rise to the formation of [(CO)₅WBr]⁻ and a bicyclo[3.2.1]octadiene (6), the Cope rearrangement product of the free divinylcyclopropane. Thermolysis of 5 affords 6 and its (CO)₅W complex. The reaction of 4 with furan (8a), 2-methylfuran (8b) and 3-methylfuran (8c) affords the (CO)₅W(bicyclo[3.2.1]oxahepta- diene) complexes (9a–c), The formation of 9a–c which is chemo-, regio- and stereospecific is explained by a tandem cyclopropanation/Cope rearrangement sequence. The bicyclic ligands 10a–c are liberated from the metal either by thermolysis of solutions of 9a–c or by addition of bromide.     

Uptake of biotin by human hepatoma cell line,Hep G2: A carrier-mediated process similar to that of normal liver

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G₂. Uptake to biotin by Hep G₂ cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na⁺ gradient-dependent as indicated by studies of Na⁺ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of ³H-biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G₂ cells with sulfhydryl group inhibitors (e.g., p-chloromer-curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G₂ cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na⁺ uptake in Hep G₂ cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na⁺-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G₂ cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work, and as such, is in the public domain in the United State of America

.     

Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine,and [11C]-and 4′-[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain

Abstract: The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and by N-demethylation, to give N-norcocaine (NC). The recent use of [N-methyl-¹¹CH₃]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O-metty/-¹¹CH₃]cocaine by methylation of the sodium salt of BE with [¹¹C]CH₃l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time-activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N-methyl-¹¹CH₃]cocaine. This strongly suggests that ¹¹C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the ¹¹C in blood plasma at 30 min was [¹¹C]CO₂ when [N-methy/-¹¹CH₃]cocaine was administered, whereas [¹¹C]CO₂ was not formed from [O-methy/-¹¹CH₃]cocaine. Only a trace of [¹¹C]NC was detected in plasma after [O-methyl-¹¹CH₃]cocaine administration. Nearly identical brain PET data were also obtained when 4′-[N-methy/-¹¹CH₃]fluorococaine and 4′-[¹⁸F]fluoro-cocaine (prepared by nucleophilic aromatic substitution from [¹⁸F]fluoride-and 4′-nitrococaine) were compared with [N-methy/-¹¹CH₃]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′-fluoroco-caine were equipotent at the dopamine reuptake site, but that 4′-fluorococaine was about 100 times more potent at the 5-hydroxytryptamine reuptake site. The studies with positron-emitting 4′-fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5-hydroxytryptamine reuptake sites to PET images taken with [N-methy/-¹¹CH₃]cocaine. [¹¹C]NC prepared by O-methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O-methy/-¹¹CH₃]cocaine. [¹¹C]BE prepared by N-methylation of norbenzoylecgonine did not show brain uptake.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Laboratory and field observations on the scuticociliate Histiobalantium from the pelagic zone of Lake Constance, FRG

Histiobalantium sp. was found regularly in the pelagic zoneof Lake Constance, FRG, over five annual cycles. Maxima of upto 6400 cells l^–1 were recorded in late summer, with similarnumbers in the 0–8 and 8–20 m depth intervals. Onan annual average, the population accounted for 10–17%of the total biomass of planktonic ciliates. In the laboratory,Histiobalantium grew well on a diet of the cryptophyte Rhodomonassp. Maximum growth rates obtained in batch cultures were 0.21and 0.33 day^–11 at 9 and 18°C, respectively. In situexperiments using diffusion chambers yielded positive growthrates in autumn and winter. The highest values recorded at theambient temperatures 5, 14 and 17°C were 0.17, 0.32 and0.40 day^–1, respectively. Comparing these results withthe different seasonal distributions and higher measured growthrates of other ciliates from Lake Constance, we conclude thatHistiobalantium is a superior competitor at relatively low algalfood concentrations. ²Present address: Fisheries & Oceans Canada, 4160 MarineDrive, West Vancouver, BC, V7V 1N6, Canada