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.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

A synaptic modification algorithm in consideration of the generation of rhythmic oscillation in a ring neural network

In consideration of the generation of bursts of nerve impulses (that is, rhythmic oscillation in impulse density) in the ring neural network, a synaptic modification algorithm is newly proposed. Rhythmic oscillation generally occurs in the regular ring network with feedback inhibition and in fact such signals can be observed in the real nervous system. Since, however, various additional connections can cause a disturbance which easily extinguishes the rhythmic oscillation in the network, some function for maintaining the rhythmic oscillation is to be expected to exist in the synapses if such signals play an important part in the nervous system. Our preliminary investigation into the rhythmic oscillation in the regular ring network has led to the selection of the parameters, that is, the average membrane potential (AMP) and the average impulse density (AID) in the synaptic modification algorithm, where the decrease of synaptic strength is supposed to be essential. This synaptic modification algorithm using AMP and AID enables both the rhythmic oscillation and the non-oscillatory state to be dealt with in the algorithm without distinction. Simulation demonstrates cases in which the algorithm catches and holds the rhythmic oscillation in the disturbed ring network where the rhythmic oscillation was previously extinguished.     

Separation of newly-synthesized RNA by organomercurial agarose affinity chromatography.

Mouse myeloma cells, MOPC-31C, were incubated in the presence of 2-thiouridine and newly-synthesized RNA which appeared to contain 2-thioUMP as a constituent was separated from preexisting RNA by affinity chromatography using organomercurial agarose as a support. Both pH and salt concentration greatly affected the specific adsorption of the newly-synthesized RNA on the column. Under optimal conditions the rate of adsorption of the newly-synthesized RNA on the column was proportional to the logarithmic concentration of 2-thiouridine in the culture medium. Furthermore, at a given concentration of 2-thiouridine in the medium, a shorter incubation period caused a reduction of the rate of RNA adsorption on the column. The molecular size distributions of both total RNA and the adsorbed fraction, synthesized during 30 min in the presence of 2-thiouridine, were similar to that of RNA synthesized in the absence of the drug.     

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.