Maximum production strategy for biodegradable copolymer P(HB-co-HV) in fed-batch culture of Alcaligenes eutrophus

A novel strategy for the maximum production of a biodegradable copolymer, poly(3-hydroxybutyric-co-hydroxyvaleric) acid, P(HB-co-HV), was developed, based on the kinetic parameters obtained from fed-batch culture experiments of Alcaligenes eutrophus. The effects of various culture conditions such as mole ratio of carbon:nitrogen in feed medium (C/N); total fatty acids concentrations; and addition ratio of fatty acids on cultivation properties such as the specific rates of cell formation, mu (h-1), P(HB-co-HV) production, rho[g.P(HB-co-HV)/g.cell/h], production yield from fatty acids [g.P(HB-co-HV)/g.fatty acid], and mole fraction of monomeric units in the copolymer [mol.(HV)/{mol.(HB) + mol.(HV)}], were investigated. When nitrogen supply was sufficient for cell growth; that is, C/N (mol.nitrogen atom/mol.carbon atom) was low, mu was high, but rho and the production yield were low, because fatty acids were used mainly for energy formation and anabolic reactions in the cells. On the other hand, when nitrogen supply was limited for cell growth-that is, C/N was high-rho was high. The highest value of rho was obtained when C/N was 75. As the mole ratio of valeric acid (VA) to butyric acid (BA) in the feed medium was increased, the mole fraction of HV units in P(HB-co-HV) increased linearly. When the ratio of BA to VA in the feed medium was kept at a constant value, but C/N was increased, the mole fraction of HV units decreased. In particular, when C/N was >12, the mole fraction of HV units decreased linearly as C/N increased. When VA was utilized as the sole carbon source and C/N was fixed at 4, P(HB-co-HV) with the highest mole fraction of HV units (67 mol%) was achieved. From these results, it was shown that both C/N and the mole ratio of BA to VA in the feed medium should be well controlled for an optimal production of P(HB-co-HV) with the desired value of the mole fraction of HV units. When the addition ratio of butyric acid was 50 wt% of total fatty acids, a maximum production strategy for P(HB-co-HV) was developed and realized experimentally, which was based on a model of the relationship between mu and rho.     

Foot formation in hydra: commitment of the basal disk cells in the lower peduncle

Foot regeneration in the freshwater hydra Pelmatohydra robusta was examined using a monoclonal antibody AE03 as a marker. This antibody specifically recognizes mucous-producing ectodermal epithelial cells in the basal disk, but not cells in the peduncle region located just above the basal disk in the foot. When the basal disk was removed by amputation at the upper or lower part of the peduncle, AE03-positive (basal disk) cells always appeared at the regenerating tip of the footless polyp approximately 12-16 h later. When a small piece of tissue was cut out from the upper or lower peduncle region, the tissue invariably turned into a smooth spherical or oblong shape within a few hours. AE03 signal appeared in these spheres variably depending on their origin: when tissue pieces were derived from the lower peduncle, the signal appeared in nearly all pieces and often covered the entire surface of the pieces within 24 h. In contrast, the signal appeared in less than 10% of pieces derived from the upper peduncle. Furthermore, the signal seldom covered more than half of the surface of these pieces. When maintained for many days, pieces derived from the upper peduncle often regenerated tentacles, whereas those from the lower peduncle seldom did. These and other observations suggest that epithelial cells in the peduncle can rapidly differentiate into basal disk cells when the basal tissue is removed. However, cells in the upper peduncle are not irreversibly committed to differentiate into basal disk cells because, when cut out as small tissue pieces, they could remain AE03 negative and become tentacle cells. In contrast, the cells in the lower peduncle apparently are irreversibly committed to differentiate into basal disk cells, as they always turned rapidly into AE03-positive cells once they were physically separated from (and freed from the influence of) the basal disk itself, regardless of the separation methods used.     

A new dipeptide,O-phosphoserylethanolamine isolated from Agkistroden blomhoffi (mamushi)

A new dipeptide was isolated from several tissues of Agkistroden blomhoffi (mamushi: a venomous snake in Japan), using ion-exchange resins and thin-layer chromatography. It was identified as O-phosphoserylethanolamine by mass spectrometry and comparison with synthetic compounds using several methods. This compound was contained in several mamushi tissues including the liver, heart, brain, bile, and muscle. The concentrations of O-phosphoserylethanolamine in the liver, brain, muscle, skin, heart, and bile were 7.17+/-3.11,16.98+/-4.25,37.37+/-7.88,37.56+/-8.97,23.93+/-6.11, and 22.21+/-5.76 micromol/g, respectively.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

Diversity,abundance, and activity of archaeal populations in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity

Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom EURYARCHAEOTA: Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family METHANOMICROBIACEAE: KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for approximately 50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.     

Synthesis of the (17R)- and (17S)-isomers of volicitin,an elicitor of plant volatiles contained in the oral secretion of the beet armyworm

Both the (17R)- and (17S)-isomers of volicitin, which is contained in the oral secretion of the beet armyworm and induces corn seedlings to emit a blend of volatile compounds to attract the natural enemy of the herbivore, were synthesized via the semi-hydrogenation of an intermediary diyne and (Z)-selective olefination as the key steps. They were both obtained as crystalline compounds.     

Synthesis of 4'-C-ethynyl and 4'-C-cyano purine nucleosides from natural nucleosides and their anti-HIV activity

Purine 2'-deoxynucleosides bearing an ethynyl or a cyano group at C-4' of the sugar moiety were synthesized from the corresponding 2'-deoxynucleosides. These compounds exhibited very potent anti-HIV activity, and remained active against drug resistant HIV strains.     

Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C

Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). The cytochalasin D-induced inhibition of SET activity was partially, but significantly, reversed by jasplakinolide, a cell permeable stabilizer of F-actin, whereas TPA-induced inhibition of SET activity was not reversed by jasplakinolide. To elucidate whether the subcellular localization of SET was changed in response to cytochalasin D or TPA, we expressed the SET fused with the green fluorescent protein (SET-GFP) in COS-7 cells and observed the subcellular distribution of SET-GFP under a confocal laser scanning fluorescent microscope. Neither cytochalasin D nor TPA markedly changed the SET-GFP cellular localization, although these drugs caused morphological change in the GFP-transfected COS-7 cells. In addition, SET activity was not altered by the treatment with either colchicine, an inhibitor of microtubule polymerization, or taxol, a stabilizer of microtubule polymerization. These results suggest that the SET uptake activity was regulated by the state of the actin cytoskeleton and that TPA exerts its inhibitory action on SET activity, in part, via disruption of F-actin and subsequent morphological change in cells.     

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.