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Ca2+clearance in frog motor nerve terminals was studied by fluorometry of Ca2+indicators. Rises in intracellular Ca2+([Ca2+]i) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca2+transient) reached a peak or plateau within 6–20 stimuli and decayed at least in three phases with the time constants of 82–87 ms (81–85%), a few seconds (11–12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca2+pumps at the cell membrane by external Li+and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li+alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca2+transients. Blocking Ca2+pumps at Ca2+stores by thapsigargin did not affect 100 stimuli-induced Ca2+transients but increased the initial decay time constant of 200 stimuli-induced Ca2+transients with no change in other parameters. Inhibiting mitochondrial Ca2+uptake by carbonyl cyanidem-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca2+transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca2+transients against [Ca2+]iyielded the supralinear [Ca2+]idependence of Ca2+efflux out of the cytosol. Blocking Ca2+extrusion or mitochondrial Ca2+uptake significantly reduced this [Ca2+]i-dependent Ca2+efflux. Thus Ca2+-dependent mitochondrial Ca2+uptake and plasmalemmal Ca2+extrusion clear out a small Ca2+load in frog motor nerve terminals, while thapsigargin-sensitive Ca2+pump boosts the clearance of a heavy Ca2+load. Furthermore, the activity of plasmalemmal Ca2+pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.

The Si(100) surfaces undergo an order–disorder phase transition from the 2×1 to the c(4×2) structure below the critical temperature Tc, which is strongly influenced near Tcby surface defects. We study the missing dimer effects on both the phase transition and the dimer buckling dynamics in the vicinity of missing dimers, based on the Ising spin model Hamiltonian. Temperature dependence of the apparent “order parameter” corresponding to the normalized (1412)low energy electron diffraction spot intensity was calculated by the Metropolis method and the buckling dynamics was calculated by the kinetic Monte-Carlo simulation. The “order parameter” is strongly suppressed near Tcas missing dimer concentration increases, while the suppression is relaxed with lowering temperature. The suppression of the “order parameter” near Tcis caused by formation of out-of-phase domain boundaries induced by missing dimers, while the out-of-phase domain boundaries are diminished as temperature decreases. From the time evolution of dimer buckling near Tc, the time averaged buckling configuration over duration Δtwas calculated in the vicinity of missing dimers. It suggests strong dependence on the scanning velocity of the scanning tunneling microscope image in the vicinity of missing dimers.

Summary.: It was observed by solution-state 13C NMR spectroscopy that a great portion of the 13C of [1-13C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-13C]Glycine was detected along with [1-13C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-13C]serine. This formation of [1-13C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state 13C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [13C]formate revealed that serine C3 was labelled specifically with 13C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine.

Abnormal CAG repeat expansion in the alpha1A voltage-dependent calcium channel gene is associated with spinocerebellar ataxia type 6, an autosomal dominant cerebellar ataxia with a predominant loss of the Purkinje cell. A reverse transcriptase-polymerase chain reaction analysis of mRNA from mouse Purkinje cells revealed a predominant expression of the alpha1A channel lacking an asparagine-proline (NP) stretch in the domain IV (alpha1A(-NP)). Human alpha1A channels carrying various polyglutamine length with or without NP were expressed in HEK293 cells, and channel properties were compared using a whole-cell voltage clamp technique. alpha1A(-NP), corresponding to P-type channel, with 24 and 28 polyglutamines found in patients showed the voltage dependence of inactivation shifting negatively by 6 and 11 mV, respectively, from the 13 polyglutamine control. Contrarily, the alpha1A channel with NP (alpha1A(+NP)), corresponding to Q-type channel, with 28 polyglutamines exhibited a positive shift of 5 mV. These results suggest that altered function of alpha1A(-NP) may contribute to degeneration of Purkinje cells, which express predominantly alpha1A(-NP), due to the reduced Ca(2+) influx resulting from the negative shift of voltage-dependent inactivation. On the other hand, other types of neurons, expressing both alpha1A(-NP) and alpha1A(+NP), may survive because the positive shift of voltage-dependent inactivation of alpha1A(+NP) compensates Ca(2+) influx.

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6J×C3H/HeJ F₁ and C3H/HeJ×C57BL/6J F₁ mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9). These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6JxC3H/HeJ F(1) and C3H/HeJxC57BL/6J F(1) mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9). These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.

Three kinds of ²H-labeled Bombyx mori silk fibroin samples (with [2,2-²H2]Gly, [3,3,3-²H3]Ala, or [2,3,5,6-²H4]Tyr) were obtained by oral administration of either the labeled amino acid or ²H2O to 5th instar larvae. The administration of ²H2O alone yielded a high degree of selective deuteration at the alanine methyl group, since the incorporation of ²H2O occurs between fumarate and malate in the tricarboxylic acid (TCA) cycle of the silk fibroin synthetic pathway. Uniaxially oriented silk fibers were prepared as samples for ²H-NMR spectroscopy. An analysis of the quadrupole echo line shape was carried out in order to determine the angle of the deuterium-labeled group relative to the fiber axis, i.e., of the Cα−²H bond vectors in glycine and of Cα−Cβ²H3 in alanine. With the fiber axis aligned parallel to the magnetic field, quadrupole splittings were obtained as 117.8 and 39.8 kHz for [2,2-²H2]Gly- and [3,3,3-²H3]Ala-labeled silk, respectively. These values are identical with those obtained from the ²H-NMR powder patterns of the unaligned samples, within experimental error. From the angular dependence of the quadrupole splittings, it was thus calculated that the Cα−²H bonds of glycine as well as the Cα−Cβ²H3 bond of alanine make an angle of approximately 90° relative to the fiber axis. These steric constraints were then used to evaluate the torsion angles, φ and ψ, for the glycine and alanine residues within the protein backbone. These data, determined independently by solid-state ²H NMR, thus verified and narrowed down the allowed region in the Ramachandran (φ, ψ) map obtained from previous solid-state ¹³C- and ¹⁵N-NMR studies.

The spermatophore of the silkmoth,Bombyx mori, is a reactor with a specific energy-yielding system for sperm maturation, the arginine degradation cascade. On mating, the highly viscous secretions from various glands in the male reproductive tract, which contain many enzymes and their substrates, are transferred to the female bursa (b.) copulatrix to form the spermatophore. In the spermatophore, transferred arginine-rich proteins are digested by initiatorin, an Arg-C endopeptidase of serine-protease type, and a carboxypeptidase. The produced free arginine is then hydrolyzed to urea and ornithine by arginase. Ornithine is metabolized to glutamate, follwed by forming alanine and 2-oxoglutarate. The latter, as a member of TCA-cycle, is a preferred respiratory substrate for spermatozoa and accelerates the post-testicular sperm maturation.

1. Application of 0.15 mM 1-(hydroxyethylamino)-2,4,6-trinitrobenzene (HEATNB) to the frog neuromuscular junction induced a marked increase (4.0-fold) in the amplitude of nerve-evoked end-plate potentials (EPPs) obtained from intracellular and extracellular records, but only a slight increase (1.9-fold) in the frequency of miniature EPPs (MEPPs) obtained from intracellular records. The effects of HEATNB on EPP amplitude and MEPP frequency showed a similar time course, reaching a plateau level approximately 40 min after the start of application and returning to the control level after wash. The difference in the effects of HEATNB on EPP and MEPP frequency suggests that it specifically enhances synchronous transmitter release. 2. Comparing the effects and structure of HEATNB with those of 2,4,6-trinitrobenzene-1-sulfonic acid, we conclude that the observed increase in transmitter release is due to the effects of the trinitrobenzene moiety of those reagents. 3. The distribution of MEPP amplitude was unchanged by HEATNB treatment, indicating that its effects are presynaptic. 4. Among four components of short-term synaptic plasticity, HEATNB greatly decreased (approximately 70%) augmentation and increased (approximately 50%) potentiation, but had little effect on fast and slow facilitations. These results suggest that each of the short-term plasticities has a different mechanism and that HEATNB affects the same mechanisms as those of augmentation. 5. Even when a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, was loaded into the presynaptic nerve terminal, the effects of HEATNB were not changed in nature, suggesting that effects of HEATNB persist independently of intracellular Ca2+ concentration. 6. These observations suggest that HEATNB may affect specific protein(s) involved primarily in synchronous transmitter release and not asynchronous release.

Immotile apyrene spermatozoa of the silkworm have a thick electron-dense flagellar membrane. On activation of apyrene spermatozoa by initiatorin, a prostatic endopeptidase of the silkworm, or by trypsin, the flagellar membrane becomes thinner, with the formation of many micropores. It then resembles that of motile apyrene spermatozoa in vivo.

Observation by electron and phase-contrast microscopy demonstrated that inBombyx morithe dissociation of eupyrene bundles, apparently through digestion of the prostatic secretion or an endopeptidase, was promoted by the vigorously flagellating movement of apyrene spermatozoa in the spermatophore.

High motility of eupyrene sperm of a grasshopper (Omocestus ventralis) was induced by cAMP. Both trypsin and cAMP were necessary for high motility of eupyrene sperm of three other species of grasshoppers. The same was found for apyrene sperm of the silkmoth,Bombyx mori, when they had been wahsed free from seminal plasma. On the other hand, in locusts (Locusta migratoria), in which the yellow gland has high Arg-C endopeptidase activity, sperm motility was induced by trypsin, like that of apyrene sperm of Lepidoptera. Thus in these two orders of Insecta, sperm motility appears to be induced by the same two-step process: the first step by Arg-C endopeptidase, and the second by cAMP.