Biochemical Characterization and Phylogenetic Analysis Based on 16S rRNA Sequences for V-Factor Dependent Members of <Emphasis Type="Italic">Pasteurellaceae</Emphasis> Derived from Laboratory Rats

Phylogenetic analysis based on 16S rRNA sequences with sequence data of some bacterial species of Pasteurellaceae related to rodents deposited in GenBank was performed along with biochemical characterization for the 20 strains of V-factor dependent members of Pasteurellaceae derived from laboratory rats to obtain basic information and to investigate the taxonomic positions. The results of biochemical tests for all strains were identical except for three tests, the ornithine decarboxylase test, and fermentation tests of D(+) mannose and D(+) xylose. The biochemical properties of 8 of 20 strains that showed negative results for the fermentation test of D(+) xylose agreed with those of Haemophilus parainfluenzae complex. By phylogenetic analysis, the strains were divided into two clusters that agreed with the results of the fermentation test of xylose (group I: negative reaction for xylose, group II: positive reaction for xylose). The clusters were independent of other bacterial species of Pasteurellaceae tested. The sequences of the strains in group I showed 99.7–99.8% similarity and the strains in group II showed 99.3–99.7% similarity. None of the strains in group I had a close relation with Haemophilus parainfluenzae by phylogenetic analysis, although they showed the same biochemical properties. In conclusion, the strains had characteristic biochemical properties and formed two independent groups within the “rodent cluster” of Pasteurellaceae that differed in the results of the fermentation test of xylose. Therefore, they seemed to be hitherto undescribed taxa in Pasteurellaceae.     

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.     

Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10 mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1 min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.     

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.     

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Construction of a tailor-made L (2S,3S)-butanediol dehydrogenase by exchanging domains between native structural analogs

The development of a stable L-BDH chimera was attempted by exchanging whole domains between two native structural analogs, L-BDH and meso-BDH, because the S-configuration specificity of L-BDH is valuable from the standpoint of its application but its activity is unstable, whereas meso-BDH is stable. The domain chimeras obtained indicated that the leaf-like structures constituting three domains were likely to be mainly associated with chiral recognition, and the fourth domain, the basic domain, is likely to be mainly associated with enzyme stability. A combination of the leaf domains of L-BDH and the basic domain of meso-BDH attained a sufficient level of practical use as an artificial L-BDH chimera, because the resulting enzyme had both stability and S-configuration specificity. However, the levels of stability and specificity were slightly lower than those of the respective enzymes from which they were derived.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.