Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

Sodium- and Calcium-Dependent Spike Potentials in the Secretory Neuron Soma of the X-Organ of the Crayfish

Membrane characteristics of neuron somata in the medulla terminalis ganglionic X-organ of crayfish have been investigated with intracellular glass microelectrodes. The soma membrane developed action potentials with 10–20 mv of overshoot. Delayed rectification appeared at 10–20 mv above resting membrane potential. In 50% of the neuron somata examined, action potentials were observed in Na-free medium or TTX medium. The peak potential level of the spike in these media depended on the extracellular concentration of Ca ion. It increased with the Ca concentration. In low calcium media, the peak potential level of the spike varied with Na concentration. Action potentials of the X-organ-sinus gland tract disappeared after bathing in Na-free or TTX medium, suggesting that the conductive action potential was dependent on Na ions. From these results, it is concluded that there are two systems in the neuron soma, one of which responds to the Na ion and the other, to the Ca ion. Inhibitory innervation of the X-organ by the cerebral ganglion was manifested by IPSP's when the optic peduncle was stimulated. A postulated connection between the Ca-dependent spike and the release of hormone in X-organ neuron somata is discussed.     

A rapid and simple method for plasmid copy number comparison

Summary A rapid, simple, and sensitive method for plasmid copy number comparison was developed. The extracted plasmids from the same amount of cells were subjected to agarose gel electrophoresis and the gels photographed. The photographs were processed by a Macintosh image analyser to enumerate the densities of plasmid bands. As a size reference, λ-DNA digested with a restriction enzyme was used. The densities divided by size of plasmids (base pair) would represent relative values of their copy numbers.     

Internal calcium concentration and potassium permeability in Paramecium

Ca or EGTA was ionophoretically injected into Paramecium tetraurelia to change [Ca]₁. Ca decreased the resting membrane resistance and hyperpolarized the membrane. EGTA had the opposite effect. EGTA following TEA, which suppress G_K, had little effect on resistance or resting potential. The I—V relation at steady state was studied before and after EGTA injection while the cell bathed in either K- or TEA-solution. The response to inward test pulses after EGTA injection was similar to that after TEA injection. These results show that [Ca]_i controls a steady-state K permeability in Paramecium tetraurelia. A prolonged Ca-spike was recorded after EGTA injection. The plateau potentials in various Ca concentrations in a TEA-solution show the Nernst slope (29 mV for tenfold change in [Ca]₀). This result suggests that the prolonged depolarization in this condition is due to a Ca current, after suppression of K-permeability and when [Ca]_i is low. The difficulty of obtaining quantitative data an the internal Ca, and the difference between the effects of EGTA injection and TEA injection are discussed.     

Constancy of rDNA Content during Dedifferentiation of Carrot and Jerusalem Artichoke Explants

When carrot explants were cultured with phytohormones, DNA synthesistook place synchronously in the explants and a satellite DNAwith a heavier density in CsCl than the bulk DNA replicatedin the earliest phase of the first replication period. The earlyreplicating carrot satellite consisted of a component havingan identical density to carrot rDNA and another component havinga density between the p-value of carrot rDNA and that of thebulk DNA. DNA-rRNA hybridization was used to explore the possibilitythat this early replication of the satellites leads to amplificationof rDNA in the explant cells, in which massive ribosome synthesisis known to occur. The results showed that there was neitheramplification nor underreplication of rRNA genes during callusformation and its growth. Experiments with explants of Jerusalem artichoke tuber, whichare well known as a synchronous replication system, showed thata component slightly heavier than the bulk DNA was synthesizedat the early phases of the first replication period. However,the density of this early replicating satellite differed fromthat of artichoke rDNA. DNA-rRNA hybridization experiments againshowed no gross changes of rDNA content during dedifferentiationof this plant system. (Received September 30, 1981; Accepted January 5, 1982)     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.