Hypnotic accumulation and hangover in elderly inpatients: a controlled double-blind study of temazepam and nitrazepam.

The hypnotic and residual sedative effects of the first and seventh of seven regular night-time doses of nitrazepam 5 mg, temazepam 20 mg, and placebo were studied in 58 elderly inpatients. Plasma temazepam and nitrazepam concentrations rose by about 50% and 113% respectively between the mornings of day 1 and day 7. Patients reported sleeping well more often after the first dose of either hypnotic (p less than 0.05), but there was no difference after the seventh dose. Reaction time was unchanged on the morning after the first dose but was significantly prolonged after the seventh dose of both hypnotics (p less than 0.01). The time taken to eliminate the letter E from a page of prose tended to be prolonged after the first dose of both drugs (temazepam v placebo, p less than 0.05; nitrazepam v placebo, not significant) and was further prolonged on the morning after the seventh dose of nitrazepam (nitrazepam v placebo, p less than 0.05). Thus plasma accumulation of the drug was associated with a deterioration in daytime performance. This change in performance did not correlate with age, cerebral blood flow, or plasma concentration, but patients of low intelligence tended to be more severely affected.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Effector-induced conformational transitions in Ascaris suum phosphofructokinase. A fluorescence and circular dichroism study.

Results of activity and spectral studies using fluorescence and circular dichroism show that AMP and fructose 2,6-bisphosphate (F-2,6-P2) activate Ascaris suum phosphofructokinase through specific and similar conformational changes. Inorganic compounds like (NH4)2SO4 and KH2PO4 also induce structural alterations in the enzyme in a manner different from those caused by AMP and F-2,6-P2. The enzyme is activated by both AMP and F-2,6-P2, in 20 mM phosphate buffer, pH 6.6, with 0.2 mM ATP and 1 mM F-6-P. The Kact values for AMP and F-2,6-P2 are 25 +/- 3 microM and 1.5 +/- 0.2 microM, respectively. Both effectors quench enzyme tryptophan fluorescence in phosphate, pH 6.6, in a concentration-dependent manner. The Kd values determined from the decrease in emission intensity at 342 nm as a function of effector concentration are 24 +/- 3 microM for AMP and 1.00 +/- 0.15 microM for F-2,6-P2, in excellent agreement with the values of Kact. Both effectors also produce dramatic changes in the CD spectrum of the enzyme, in the region from 240 to 190 nm representing the peptide backbone. Secondary structure calculations suggest an increase in the alpha-helical content of the enzyme in the presence of either effector. The Kd values obtained from the concentration dependence of the decrease in ellipticity at 210 nm are 22.8 +/- 5.3 microM and 1.3 +/- 0.2 microM, respectively, for AMP and F-2,6-P2, once again in close agreement with the Kact values for these effectors. The data imply that activation of phosphofructokinase by these effectors is concomitant with structural changes in the enzyme. Further, comparison of the difference CD spectra for the effects of AMP and F-2,6-P2 show that both of them produce similar conformational changes and probably stabilize a similar final activated state of the enzyme. Other hexose phosphate analogues such as fructose 6-phosphate, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate do not affect the CD spectrum of the enzyme. Ammonium sulfate has no effect on the CD spectrum of the enzyme in phosphate buffer but does cause a significant alteration in the spectrum obtained in Mes. Gel filtration high performance liquid chromatography using a Borosil TSK 400 column shows that the tetrameric state of the native enzyme is not affected by the presence of the effectors.     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.

A new property of ursolic acid, lipoxygenase and cyclooxygenase inhibition, has been described in an acetone-extract of heather flowers (Calluna vulgaris) which could help explain the anti-inflammatory characteristics of this plant. In mouse peritoneal macrophages, human platelets and differentiated HL60 leukemic cells, ursolic acid, at 1 microM, blocks arachidonate metabolism.     

Complexation with heparin prevents adhesion between fibrin-coated surfaces.

Heparin, in Langmuirian fashion, binds stoichiometrically with high affinity, Kd approximately 100 nM, to both fibrinogen and fibrin adsorbed as monomolecular films to lecithin-coated, microscopic, polystyrene-divinylbenzene beads. Complex formation inhibits aggregation of fibrin-coated beads, and it also results in dissociation of preformed aggregates of fibrin-coated beads. These phenomena are not caused by desorption of fibrin(ogen), indirect inhibition of thrombin activity, or mere electrostatic repulsion of charged particles. Instead, these data are consistent with the proposal that the complexed heparin interferes directly with dimer formation between fibrin molecules adsorbed to colliding beads. We describe these phenomena and their application to the development of sensitive analytical methods for quantitating heparin. Based on these observations, we also propose a role for endogenous heparin in the physiologic regulation of fibrin-mediated adhesion of surfaces.     

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.