Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

Some Physical and Chemical Properties of Nuclease P1

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP₅₀) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

Existence of Sixteen 2′→5′ Dinucleotides in Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Sixteen 2′→5′ dinucleotides; (2′–5′)pA-A, pA-G, pA-C, pA-U, pG-A, pG-G, pG-C, pG-U, pC-A, pC-G, pC-C, pC-U, pU-A, pU-G, pU-C, and pU-U were detected in nuclease P₁ digest of a technical grade yeast RNA by means of gel filtration on Sephadex G-10, DEAE-Sephadex A-25 column chromatography in the presence of 7 m urea, paper electrophoresis and paper chromatography. Content of each dinucleotide was about 0.1 to 0.6% of the digest. As the sixteen 2′→5′ dinucleotides were found in all of the digests of technical grade RNA preparations tested, each polynucleotide chain in the preparations may be concluded to contain several per cent of the 2′–5′ minor phosphodiester linkages in addition to the 3′–5′ major phosphodiester linkages.     

Occurrence of Nuclease P1-Malonogalactan Complex

Nuclease P₁ from Penicillium citrinum was found to be produced in a form of complex with malonogalactan (a galactan, 1, 5-β-galactofuranoside polymer esterfied with malonic acid at position 3) in the culture on wheat bran. Neither nuclease P₁-malonogalactan complex nor malonogalactan was produced in a liquid medium. Nuclease P₁-malonogalactan complexes, P₁-MG I, II, and III were purified from an aqueous extract of the culture on wheat bran. The most anionic complex, P₁-MG III, was composed of the protein, carbohydrate and malonic acid in the ratio of 1: 2.6: 0.5 (w/w). The complex was not dissociated by purification procedures including fractionations with acetone and ammonium sulfate, gel filtration and DEAE-cellulose chromatography. A malonogalactan-specific carboxylesterase was found in culture of the same mold on wheat bran. Nuclease P₁-malonogalactan was demalonylated by the esterase to yield nuclease P₁-galactan. The binding of nuclease P₁ to galactan was rather loose so that nuclease P₁-galactan complex was partially dissociated by DEAE-cellulose chromatography. Attempt to reconstitute the complex from nuclease P₁ and malonogalactan upon mixing was unsuccessful. Exogenously supplemented nuclease P₁ did not associate with malonogalactan in the growing culture on wheat bran, either.

Several extracellular enzymes such as RNase, β-galactosidase and protease were also found in a form of complex with malonogalactan in the culture on wheat bran.     

Brain regeneration in anuran amphibians

Urodele amphibians are highly regenerative animals. After partial removal of the brain in urodeles, ependymal cells around the wound surface proliferate, differentiate into neurons and glias and finally regenerate the lost tissue. In contrast to urodeles, this type of brain regeneration is restricted only to the larval stages in anuran amphibians (frogs). In adult frogs, whereas ependymal cells proliferate in response to brain injury, they cannot migrate and close the wound surface, resulting in the failure of regeneration. Therefore frogs, in particular Xenopus, provide us with at least two modes to study brain regeneration. One is to study normal regeneration by using regenerative larvae. In this type of study, the requirement of reconnection between a regenerating brain and sensory neurons was demonstrated. Functional restoration of a regenerated telencephalon was also easily evaluated because Xenopus shows simple responses to the stimulus of a food odor. The other mode is to compare regenerative larvae and non-regenerative adults. By using this mode, it is suggested that there are regeneration-competent cells even in the non-regenerative adult brain, and that immobility of those cells might cause the failure of regeneration. Here we review studies that have led to these conclusions.     

Sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger

In this work, we examined the antioxidant role of mucin, a typical sialic acid containing high-molecular weight glycoprotein. The function of mucin as a hydroxyl radical (.OH) scavenger was characterized using bovine submaxillary gland mucin (BSM). Non-treated BSM effectively protected DNA from the attack of .OH; however, desialylated BSM lost this potential. Moreover, we estimated the scavenging effects of BSM against .OH generated by UV irradiation of hydrogen peroxide using ESR analysis. Our results indicate that BSM has .OH scavenging ability the and sialic acid in mucin is an essential moiety to scavenge .OH.     

Self-Incompatibility Involved in the Level of Acetylcholine and cAMP

Elongation of pollen tubes in pistils after self-pollination of Lilium longiflorum cv. Hinomoto exhibiting strong gametophytic self-incompatibility was promoted by cAMP and also promoted by some metabolic modulators, namely, activators (forskolin and cholera toxin) of adenylate cyclase and inhibitors (3-isobutyl-1-methylxanthine and pertussis) of cyclic nucleotide phosphodiesterase. Moreover, the elongation was promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, L-α-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. A potent inhibitor (neostigmine) of acetylcholinesterase (AChE) as well as acetylcholine also promoted the elongation. cAMP enhanced choline acetyltransferase (ChAT) activity and suppressed AChE activity in the pistils, suggesting that the results are closely correlated with self-incompatibility in L. longiflorum. In short, it came to light that cAMP modulates ChAT (acetylcholine-forming enzyme) and AChE (acetylchoine-decomposing enzyme) activities to enhance the level of ACh in the pistils of L. logiflorum after self-incompatible pollination. These results indicate that the self-incompatibility on self-pollination is caused by low levels of ACh and/or cAMP.Key Words: pollen tubes, self-incompatibility, Lilium longiflorum, cAMP, acetylcholie, AChE, ChATCyclic AMP (cAMP) is an essential signaling molecule in both prokaryotes and eukaryotes.¹ The existence of cAMP in higher plants was questioned by some reviewers^2–4 in the mid 1970''s, so that many workers were discouraged from studying roles in plant biology. However, its presence was confirmed by mass spectrometry⁵ and infrared spectrometry⁶ in the early 1980''s and increasing evidence^7–12 now suggests that cAMP makes important contributions in plant cells, as in animals.Lily (Lilium longiflorum) exhibits strong gametophytic self-incompatibility.^13,14 Thus, elongation of pollen tubes in the pistil after self-incompatible pollination in L. longiflorum cv. Hinomoto stops halfway, in contrast to the case after cross-compatible pollination (cross with cv. Georgia).¹⁴ This self-incompatibility appears to be associated with the stress and self-incompatible pollination on stigmas of lilies results in activation and/or induction of enzymes such as NADH- and NADPH-dependent oxidases, xanthine oxidase, superoxide dismutase (SOD), catalase and ascorbate peroxidase in the pistils.¹⁵ The activities of NADH- and NADPH-dependent oxidases (O₂⁻-forming enzymes), however, are known to be suppressed by cAMP¹⁶ and increase in the level of cAMP in guinea pig neutrophils results in their decreased expression.¹⁷ The level of O₂⁻ reactions with SOD is also decreased by cAMP.¹⁸ In the case of the lily, inhibition of NADH- and NADPH-dependent oxidases by cAMP was found to be noncompetitive with NAD(P)H.¹⁶ We hypothesized that decrease in active oxygen species such as O₂⁻ and suppression of stress enzyme activities in self-pollinated pistils of lily by cAMP might cause elongation of pollen tubes after self-pollination and this proved to be the case. Namely, elongation of pollen tubes after self-incompatible pollination in lily was promoted by exogenous cAMP at a concentration as low as 10 nM, a conceivable physiological level.¹³ Moreover, similar elongation could be achieved with adenylate cyclase activators [forskolin(FK) and cholera toxin] and cAMP phosphodiesterase inhibitors [3-isobutyl-1-methylxanthine (IBMX) and pertussis toxin].^14,19 These phenomena led us to examine the involvement of endogenous cAMP in pistils after self-incompatible or cross-compatible pollination. As expected, the level of endogenous cAMP in pistils after self-pollination was approximately one half of that after cross-pollination. Furthermore, this was associated with a concomitant decrease in adenylate cyclase and increase in cAMP phosphodiesterase.¹⁹Many researchers in the field of plant biology have been unsuccessful in attempts to estimate the quantity of cAMP and to detect activities of adenylate cyclase and cAMP phosphodiesterase. On major difficulty is the presence of proteases and we have overcome this problem by using protease inhibitors, such as aprotinin and leupeptin.¹⁹In 1947, acetylcholine (ACh) of higher plants was first reported in a nettle (Urtica urens) found in the Himalaya mountain range.²⁰ In 1983, its existence in plants was confirmed by mass spectrometry of preparations from Vigna seedlings.²¹ In our preliminary studies, CCC (chlorocholinechloride), a plant growth retardant (specifically an anti-gibberellin), enhanced the elongation of the pollen tubes in pistils after self-incompatible pollination in lilies. This led us to investigate whether other choline derivatives cause similar effects and positive findings were obtained with ACh, acetylthiocholine and L-α-phosphatidlylcholine.²² Moreover, the elongation was also promoted by neostigmine, an inhibitor of acetylcholine esterase (AChE) activity. In line with these results, choline acetyltransferase (ChAT) demonstrated low and AChE high activity in pistils after self-incompatible pollination.The positive influence of cAMP^14,19 and ACh²² in pistils of L. longiflorum after self-incompatible pollination encouraged us to examine the involvement of these two molecules in regulation of pollen tube elongation of lily after self-incompatible and cross-compatible pollination. As a result, it was revealed that cAMP promotes ChAT and suppresses AChE activity in pistils after both self- and cross-pollination. In other words, the self-incompatibilty in pistils of L. longiflorum appears to be due to levels of ACh and/or cAMP below certain threshold values.Hitherto, these substances have not been recognized to play important roles in the metabolic systems of higher plants. However, given their conservation through evolution, it is natural that such central metabolic substances make essential contributions, regardless of the organism. We have succeeded in establishing physiological functions of cAMP and ACh in pistils of lily^14,19,22 and this points to use of plant reproductive organs such as research materials. The exact responsibilities of the two molecules may depend on differences in tissues or organs of plants and further molecular biological studies in this area are clearly warranted. This issue is currently being investigated.     

Regulation of ammonia homeostasis by the ammonium transporter AmtA in Dictyostelium discoideum

Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold Dictyostelium discoideum; however, it is largely unknown how intracellular ammonia levels are controlled. In the Dictyostelium genome, there are five genes that encode putative ammonium transporters: amtA, amtB, amtC, rhgA, and rhgB. Here, we show that AmtA regulates ammonia homeostasis during growth and development. We found that cells lacking amtA had increased levels of ammonia/ammonium, whereas their extracellular ammonia/ammonium levels were highly decreased. These results suggest that AmtA mediates the excretion of ammonium. In support of a role for AmtA in ammonia homeostasis, AmtA mRNA is expressed throughout the life cycle, and its expression level increases during development. Importantly, AmtA-mediated ammonia homeostasis is critical for many developmental processes. amtA(-) cells are more sensitive to NH(4)Cl than wild-type cells in inhibition of chemotaxis toward cyclic AMP and of formation of multicellular aggregates. Furthermore, even in the absence of exogenously added ammonia, we found that amtA(-) cells produced many small fruiting bodies and that the viability and germination of amtA(-) spores were dramatically compromised. Taken together, our data clearly demonstrate that AmtA regulates ammonia homeostasis and plays important roles in multiple developmental processes in Dictyostelium.