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.     

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro , furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli , purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.     

Relationships among the three light-induced absorbance changes related to the reaction-center of photosystem II

The relationships among X₅₉₁, Cyt-b₅₅₉ and C-550 in the primaryphotoact of PS-II were analysed by examining the effects ofvarious inhibitory substances and treatments on the light-inducedabsorbance changes of these components. The results were fully explainable by the scheme previouslypresented by Huzisige, in which two photoreactions are involvedin PS-II. Our conclusion is that X₅₉₁ acts as the electron acceptorfor one of the photoreactions in PS-II. (Received October 23, 1978; )     

A light and electron microscopic study of the flagellate-to-ameba conversion in the MyxomyceteStemonitis pallida

Summary The flagellate-to-ameba conversion process of the MyxomyceteStemonitis pallida was investigated with Nomarski optics and electron microscopy. The flagellate has two flagella, a long and a short one. When the water film containing the flagellates becomes very thin, they retract their flagella, usually the short one first and then the long one. The short flagellum is retracted by only one method, in which the sheath membrane of the flagellum fuses with the cell membrane, consequently causing the axoneme to be absorbed into the cytoplasm. Retraction of the long flagellum can be divided into four types. In all cases, fusion of the sheath membrane and the cell membrane takes place. The retracted axoneme of the long flagellum sometimes beats convulsively for about 10 minutes after retraction, and after 10–15 minutes it became indistinguishable as it was detached from the blepharoplast.Analysis of thin sections shows that the retracted axonemes disintegrate in the following squence: B-tubules, A-tubules, spokes, central microtubules. In almost all cells the degradation begins immediately after retraction and is completed within 90 minutes. Only on rare occasions, structures which seem to have been derived from retracted axonemes are observed in the ameba about 90 minutes after conversion. The basal bodies and cytoplasmic microtubules are a little more stable than the retracted axonemes. Some basal bodies of the short flagellum, whose C-tubules are affected, are present in the amebae more than 90 minutes after conversion. Cytoplasmic microtubules decrease in number and become shorter in the amebae after about 24 hours, when newly formed regions filled with flocculent material appear.     

Resolution of anomeric 2-amino-2-deoxy-d-glycofuranosides and -glycopyranosides by cation-exchange chromatography

Methyl 4,6-O-benzylidene-2-deoxy-α-D-ribo-hexopyranoside (1) is converted into methyl 3,4-di-O-benzoyl-6-bromo-2,6-dideoxy-α-D-ribo-hexopyranoside (3) via the 3-O-benzoyl derivative (2) of 1 by subsequent treatment with N-bromosuccinimide. Compound 3 is the key intermediate in high-yielding, preparative syntheses of the title dideoxy sugars, which are constituents of many antibiotics. Dehydrohalogenation of 3 affords the 5,6-unsaturated glycoside 7. which undergoes stereospecific reduction by hydrogen with net inversion at C-5 to give methyl 3,4-di-O-benzoyl-2,6-dideoxy-β-L-lyxo-hexopyranoside (8), whereas reductive dehalogenation of 3 provides the corresponding D-ribo derivative 4. The unprotected glycosides 9 (L-lyxo) and 5 (D-ribo) are readily obtained by catalytic transesterification, and mild, acid hydrolysis gives the crystalline title sugars 10 (L-lyxo) and 6 (D-ribo) in 45 and 57% overall yield from 1 without the necessity of chromatographic purification at any of the steps.     

Calmodulin antagonists inhibit Ca2+ uptake of mitochondria of guinea pig peritoneal macrophages

The Ca²⁺ uptake of the mitochondria of guinea pig peritoneal macrophages was not stimulated by the addition of calmodulin. However, calmodulin antagonists, both phenotiazines and N-naphthalenesulfonamides, in low concentrations inhibited the Ca²⁺ uptake of the mitochondoria, as compared to the inhibition of the calmodulin-dependent stimulation of brain phosphodiesterase. These calmodulin antagonists appear to have severe side effects on active processes of the mitochondria and which are unrelated to the specific effect on calmodulin.     

ATP-dependent Ca2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca²⁺ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{4 × 10}{}^6$ macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10⁶ M^?1. In the absence of sodium azide, the value of Ca²⁺ uptake of saponin-treated macrophages was 95 nmoles/4 × 10⁶ macrophages, and its affinity constant for Ca²⁺ was 3 × 10⁵ M^?1. Saponin-treated macrophages may be suitable for the studies of Ca²⁺ transport systems in the intracellular membranes.