Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

The effects of short-chain fatty acids on the neuronal membrane functions ofHelix pomatia. I. electrical properties

The effects of short-chain fatty acids on the membrane excitability, current-voltage (I-V) characteristics, and cell volume of Helix pomatia neurons were studied. 2-Decenoic acid (DA), having 10 carbon atoms in the hydrocarbon chain, suppressed the excitability of bursting neurons RPa1 (Sakharov and Salanki, 1969) for 30-60 min, while valeric acid (VA), having 5 carbon atoms, had no significant effect on excitability. DA had three different effects on the excitability of beating neurons: in some neurons DA suppressed excitability as in bursting neurons; in a second type of neuron DA had a negligible effect on excitability; and in the neuron located near RPa1 DA had a pentylentetrazol (PTZ)-like effect, i.e., it converted the discharge of the neuron from beating to bursting. DA decreased the peak value of the current, inducing a negative-resistance region in the I-V curve of the bursting neuron without any change in the level of the voltage at which the current reaches its maximal value. DA inhibited the hyperpolarization induced by activation of the Na+ pump, tested after preliminary enrichment of neurons with Na+ ions by incubation in a potassium-free solution for 20 min. DA caused a swelling of the neuron by about 10% which was independent of the Na+ pump. In all the above-mentioned cases VA had no significant effect.     

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca²⁺ concentration. © 1993 John Wiley & Sons, Inc.     

New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF-releasing factor in serum.

The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators.     

Formation of catharanthine,akuammicine and vindoline in Catharanthus roseus suspension cells

Catharanthine and akuammicine, together with ajmalicine and strictosidine, were isolated from a culture strain of Catharanthus roseus suspension cells. The biosynthetic capability of the cultured cells to produce akuammicine, catharanthine and vindoline was confirmed by feeding experiments with dl-tryptophan-[3-¹⁴C] to yield the radioactive alkaloids.