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Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.

For corals that establish symbioses with dinoflagellate Symbiodinium spp. at the lar- val stage or later through horizontal transmission, the ecological significance of the early uptake of algal symbionts remains unknown. It has been hypothesized that early uptake of symbionts is an advantage for long-distance dispersal. Here, we tested the hypothesis that early acquisition of symbionts enhances post-settlement survival. We used a cultured strain of clade A Symbiodinium that was isolated from wild Acropora spat as the algal symbiont. Symbiotic and aposymbiotic Acropora larvae were prepared in the laboratory and settled on experimental plates in the field. The survival of settlers was monitored for 15 mo. Our results showed that more larval-stage set- tlers harbouring symbionts survived than those without, even when there was no difference in the initial density of settled larvae. We analysed the Symbiodinium clades harboured by the corals at 1 mo after settlement, and found that clade A was less abundant in the corals that grew from aposymbiotic larvae than in those that developed from symbiotic larvae. There was also a marked difference in coral survival between aposymbiotic and symbiotic larvae over this period. The higher survival rate of 'early uptake' corals was more pronounced on shaded plates. These results suggest that the early uptake of specific symbionts enhances post-settlement survival in dark places such as reef crevices, which are sites commonly settled by coral larvae.

In situ larval seeding is a low-cost technique that is currently under development for the large-scale restoration of coral populations. One problem that still needs to be solved is the preparation of coral larvae for seeding, i.e., how many larvae are required to restore a certain area? In this study, we focused on the relationship between the numbers of larvae, settlers, and survivors for three months post-settlement to determine the optimal larval seeding density. A comparison of three different larval densities (low, middle, and high) indicated that the number of settlers was proportional to the larval density, suggesting that settler density is determined by the number of larvae supplied. However, the survival rate of settlers on high-density plates was much lower than the corresponding rates on low- or middle-density plates during the first month after settlement. Moreover, most of the seeded corals had not survived on the low-density plates at three months after settlement. Therefore, the middle larval density (i.e., 5000 larvae m−2) appears to be optimal for seeding on grid plates.

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1–4 and GlcNAc α1–4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33, 43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins. Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin nor hyaluronic acid. AS antibodies did not bind to heparan sulfate or heparin, but bound to 6-O-desulfated, N-desulfated and re-N-acetylated heparin to varying degrees. ACH55 bound to tri-desulfated and re-N-acetylated heparin but hardly bound to other modified heparins. NAH antibodies did not bind to heparin and modified heparins but bound to heparan sulfate to varying degrees. NAH43 and NAH46 also bound to partially N-de-acetylated N-acetyl-heparosan. Immunohistochemical analysis in rat cerebella was performed with the antibodies. While NAH46 stained endothelia, where heparan sulfate is typically present, neither ACH55 nor AS25 stained endothelia. On the contrary ACH55 and AS25 stained the molecular layer of the rat cerebella. Furthermore, ACH55 specifically stained Purkinje cells. These results suggest that there is unordinary expression of IdoA2S-GlcNAc and IdoA-GlcNAc in specific parts of the nervous system.

The enzymatic characterization of GDP- d -mannose 3″,5″-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli . Reaction products from GDP- d -mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP- l -galactose and GDP- l -gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD + in contrast to the case of GME from Arabidopsis thaliana . This difference in the effect of NAD + on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K m and k cat were determined to be 1.20 × 10 −5 M and 0.127 s −1 , respectively, in the presence of 20 μM NAD + . The fractions of GDP- d -mannose, GDP- l -galactose and GDP- l -gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.

To clarify the pathogenesis of altered bone metabolism in diabetic state and its underlying mechanisms, the bone mineral content and fasting levels of serum intact parathyroid hormone (i-PTH), intact osteocalcin (i-OC), tartrate-resistant acid phosphatase (TRAP) and osteoclastgenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were measured in male type 2 diabetic patients and their age-matched controls. In addition, urine levels of osteoclastic markers, C-telopeptide of type I collagen (CTx), deoxypyridinoline (DPD), and N-telopeptide of type I collagen (NTx) were simultaneously determined. Serum levels of i-PTH and i-OC in diabetic patients were significantly lower than those in the controls. Conversely, serum concentrations of TRAP were significantly elevated in diabetic patients. However, no clear correlation was observed between serum i-OC and TRAP. It was also observed that urinary excretion of CTx, DPD, and NTx was significantly increased in the diabetics as compared with the controls. Unexpectedly, serum levels of OCIF/OPG tended to be higher in the diabetic group, and these values exhibited a significantly positive correlation with those of serum TRAP. There was found a significantly negative correlation between serum TRAP and bone mineral density (BMD) and also between serum OCIF/OPG and bone mineral density. It seems probable that OCIF/OPG has a suppressive role on the increased bone resorption to prevent further loss of the skeletal bone mass in type 2 diabetic patients.

A novel method, based on quantitative PCR and amplified fragment length polymorphism was applied to the analysis of high-coverage gene expression profiles during the development of rice seeds. This represents the first report of the application of this method to plants, which permitted the detection and analysis of approximately 70% of all the genes that are expressed in rice. The method was used to compare gene expression at different developmental stages, subspecies or cultivars, and phyletic lines to identify genes of interest through differences in their level of expression. Using this approach, even novel anonymous genes could be detected. Examples of these include the soluble starch synthase (SS) II-I and the rice branching enzyme 4 (rbe4) genes in the starch synthesis pathway. A profiling database was compiled and the results compared with public data on full-length cDNA sequences of rice. The method enables candidate novel genes to be immediately identified among the large numbers of genes that are expressed during the development of rice seeds. Our results will contribute to a better understanding of comparative transcriptomics in all plant species.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.

The viper dogfish, Trigonognathus kabeyai is further described on the basis of 19 male [176–470 mm in total length (TL)] and 13 female (325–539 mm TL) specimens collected from the Kumano-nada Sea. In addition, the holotype, paratype, and seven specimens collected from the stomachs of Taractichthys steindachneri and Thunnus obseus from the Ogasawara Islands, Japan, were also examined. This is the first description of large (adult) specimens. The allometric size-on-size equation Y = aX b was used to determine morphometric growth patterns, positive allometry, negative allometry, and isometry. Size at maturity was about 430 mm TL for males and about 520 mm TL for females. Myctophid fishes consisting of Diaphus gigas, Diaphus spp., Benthosema fibulatum, and Benthosema sp. were the only identifiable prey in the stomachs of Trigonognathus kabeyai examined. Most of the prey fishes were found almost intact and usually had puncture holes made by teeth. On the basis of stomach content and jaw morphology, this species is thought to be a ram feeder.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.

The cell wall architecture of bamboo at various developmental stages of culm growth was investigated by rapid-freezing and deep-etching (RFDE) electron microscopy. The unlignified primary wall (ULP) was characterized by the narrow spacing between the cellulose microfibrils in fibres, but not in parenchyma cells. The unlignified secondary wall (ULS) largely consisted of dense cellulose microfibrils with narrow spacing or "slit-like" pores. However, such pores are difficult to observe in the lignified secondary wall (LS) of fibres. The cell wall architecture of the delignified secondary wall (DLS) in fibres showed porosity similar to that of ULS. Pores in the middle lamella and secondary walls of ULS in fibres were reduced considerably or disappeared immediately after lignification. However, the pores reappeared following delignification. These findings suggest that deposition of lignin in ULS immediately proceeds in the pores during maturation to LS. Based on RFDE electron microscopic images, the pore sizes of primary and secondary fibre walls were significantly smaller in bamboo than in either Eucalyptus or Pinus, suggesting a denser arrangement of cellulose microfibrils in bamboo fibre walls than in either tree species. The author hypothesizes that the narrow spacing between cellulose microfibrils in bamboo fibres may be one of the reasons for the deposition of less lignin in bamboo than in tree species. This is the first report on the three-dimensional architecture of unlignified and lignified walls in bamboo.

Markedly damaged stands have been observed in Siebold's beech (Fagus crenata) forests on the Tanzawa Mountains, Japan, which are located close to densely populated and heavily industrial areas. We measured the delta(13)C records for the past 50 years (1944-1997) in wood cellulose sampled from relatively healthy trees in declining forests on Mt. Hinokiboramaru (1600 m). This may provide information on the history of stresses related to environmental changes, which have caused the decline symptoms. The results showed that, for all of the trees studied, wood delta(13)C has decreased with time. Also, the difference in delta(13)C among trees grew abruptly after the mid-1960s, which almost coincides with the time when the decline symptoms were markedly observed. Some trees with large reduction of wood delta(13)C exhibited the strong decreases in radial growth. This suggests that the reduction of tree growth may have been more greatly influenced by decreasing carboxylation rate than by stomatal limitation. It is unlikely that water stress and SO(2) and O(3) stresses have induced the growth reduction, because those stresses cause increasing wood delta(13)C. This is supported by the facts that wet conditions and relatively low SO(2) and O(3) levels have been observed near Mt. Hinokiboramaru. In addition, analyses of wood Ca showed no evidence that acid fog and soil acidification have affected the wood delta(13)C and growth through effects on the nutrient uptake of trees. However, what type of stresses have induced the large reduction of wood delta(13)C and growth for some of the trees studied remains unknown because of the lack of sufficient data for evaluation. In contrast, lesser reduction of wood delta(13)C from the other trees may be related to an increase in the plant water-use efficiency with increasing atmospheric CO(2) concentration.

Marked damage to Japanese fir (Abies firma) forests have been observed on Mt. Oyama, Tanzawa Mountains, Japan, which is located close to densely populated and heavily industrial areas, since the 1960s. The authors evaluated possible causes for the decline based on the {delta}{sup 13}C records of annual growth rings sampled from trees without visual decline symptoms. For comparison, the {delta}{sup 13}C records for Japanese Cedar (Cryptomeria japonica) on Mt. Oyama for which decline has not been observed were also investigated. The results showed that the {delta}{sup 13}C of rings from both tree species increased abruptly during the 1960s and then decreased after reaching a maximum value in the early 1970s, while the tree growth inversely changed with {delta}{sup 13}C. The historical changes of {delta}{sup 13}C of rings and tree growth were correlated with atmospheric SO{sub 2} concentrations with a delay of 5--10 years in their maximum responses. Exposure to SO{sub 2} at the levels estimated during the 1960s on Mt. Oyama can induce the increase of {delta}{sup 13}C of rings and the reduction of tree growth with stomatal closure being the probable cause. Previous studies have shown that Japanese fir has a lower tolerance to SO{sub 2} as compared tomore » Japanese cedar. These results suggest that the fir forests on Mt. Oyama were injured mainly by SO{sub 2} pollution during the 1960s causing the decline.« less

Localization of xyloglucan in cell walls regenerated from tobacco protoplasts {Nicotiana tabacum L.; cv. BY2) is visualized by rapid-freezing and deep-etching (RFDE) electron microscopy coupled with immunogold electron microscopy. Xyloglucan was already deposited in the cell wall 3 h after culture initiation. Xyloglucan was mainly localized along microfibrils with a lesser amount in intersections between two crossed microfibrils in 120-hour-old cells. These data support the previous hypothesis of Keegstra et al. (1973) that propose an interconnection between xyloglucan and cellulose.

A method using δ13C records of annual growth rings has been proposed for assessing environmental stresses in mature forest trees. First, we calculate the change with time in the difference (Ca-Ci) between the CO2 concentration in the atmosphere and the leaf intercellular spaces from the δ13C records in tree cellulose based on a carbon isotope fractionation model for C3 plants. The Ca-Ci value is dependent primarily on the mesophyll capacity for photosynthesis and the supply of CO2 through the stomata. Next, stomatal limitation of photosynthesis is evaluated from the ratio of ring area to Ca-Ci which is possibly indicative of leaf conductance. These parameters for the trees from a declining grove of Japanese cedar (Cryptomeria japonica) in the Kanto Plain, Japan were suggested to respond sensitively to the changes in air pollution (mainly SO2) and/or dry atmospheric conditions. This method offers a potentially useful research tool for evaluating possible causes for forest decline.

A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.

Mouth guards were used by top athletes to study the changes in muscle strength. As a result, it was found that athletes that tended to bite when exerting strength experienced an increase in muscle strength. On the other hand, the muscle strength in athletes that did not bite when exerting strength tended to decrease. The effects varied depending on to the individual and the type of athletic activity, and the results varied, i. e., some athletes experiencing an increase in maximum strength, some an increase in average strength and some an increase in work strength. Thus it was concluded that there were individual differences in terms of the strength results.

The initial factors that cause a decline in the survival of in situ settled corals remain poorly understood. In this study, we demonstrated through field experiments that the design of artificial grid plates may influence the initial survival of Acropora corals, with narrower grids being the most effective. In fact, grid plates with a 2.5-cm mesh presented the highest recorded survival rate (14%) at 6 months after settlement (representing approximately 50 corals per 0.25 m2 of plate). This is the first study where such high survival rates, matching those of cultures under aquarium conditions, were obtained in the field without using additional protective measures, such as guard nets against fish grazing after seeding. Therefore, our results provide a foundation for establishing new and effective coral restoration techniques for larval seeding, in parallel to clarifying the details of the early life stages of reef-building corals.

Recent studies have revealed that altered mineral and vitamin D metabolism is observed in diabetic patients with the complication of osteopenia. In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) on calcium homeostasis in diabetes, we have measured the serum level and urinary excretion of PTHrP as well as other serum calcium-regulating hormones in 106 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 43 control subjects. The serum concentration of intact PTH was 2.34±0.13 (mean±sem) pmol/l in NIDDM patients, which is significantly lower than the value of 3.11±0.14 pmol/l in the controls (p<

Lagunamycin, a novel 5-lipoxygenase inhibitor, was isolated from the culture broth of Streptomyces sp. AA0310. This compound showed potent rat 5-lipoxygenase inhibitory activity (IC50 6.08 microM) without lipid peroxidation.

Three new 5-lipoxygenase inhibitors, designated as BU-4601 A, B and C, were found in the fermentation broth of Streptomyces sp. strain No. AA2807. Their structures were identified as isodecyl, isoundecyl and isolauryl esters of 5-hydroxyanthranilic acid, respectively. Based on their structures, five related esters were synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase. Both naturally-occurring and chemically-synthesized compounds exhibited almost equal levels of 5-lipoxygenase inhibitory activities in vitro.

Ten metabolites produced by 4 mutants derived from Actinomadura verrucosospora subsp. neohibisca E-40, a high pradimicins producer, were isolated and their structures were determined. Strain JN-219 produced 3 novel analogs of the pradimicin A aglycone, i.e. 11-O-demethyl-7-methoxypradinone II and 11-O-demethylpradinones I and II together with a known aglycone analog, pradinone I, while the metabolites from strain JN-47 were determined to be 2 new aglycone analogs, 11-O-demethylpradimicinone I and 11-O-demethyl-7-methoxypradimicinone II and a known aglycone analog, 11-O-demethylpradimicinone II (11dM-PMN II). Products of strain JN-207 were identified as 11-O-demethyl-6-deoxypradinone I and 11dM-PMN II. Interestingly, a new pradimicin analog, 7-hydroxypradimicin A was isolated from strain JN-58 together with a new aglycone analog, pradimicinone II and 11dM-PMN II. None of these metabolites showed antifungal activity.

The biosynthetic pathway of the pradimicin-benanomicin family of antibiotics was investigated by using sinefungin and blocked mutants derived from Actinomadura verrucosospora subsp. neohibisca E-40 (a high pradimicin producer) or Actinomadura sp. AB1236 (a benanomicin producer). Addition of sinefungin to strain E-40, pradimicin A aglycone-producing mutant or strain AB1236 inhibited the formation of 11-O-demethyl-7-methoxypradinone II (11dM-7M-PN II), resulting in the accumulation of 11-O-demethylpradimicinone II and pradimicinone II. By feeding pradimicin A aglycone and its analogs to mutants blocked early in pradimicin or benanomicin biosynthesis, the following results were obtained: 11-O-demethylpradinone II, 11dM-7M-PN II 11-O-demethylpradinone I, 11-O-demethylpradimicinone I and pradimicinone I were converted to pradimicin A or benanomicin A; the remaining 6 aglycone analogs were not incorporated into the antibiotics. Pradimicin B, dexylosylpradimicin C and dexylosylbenanomicin A were converted to pradimicin A, pradimicin C and benanomicin A, respectively. A biosynthetic pathway for the antibiotics is proposed.

In order to investigate the underlying mechanism of alterations in bone mineral metabolism in patients with type 2 diabetes, we determined circulating levels of bone functional markers along with urinary excretion of sorbitol (SOR) and bone mineral density (BMD), and also examined their mutual interrelationship. A total of 151 male type 2 diabetic patients were examined in this study. Forty-eight age-matched male healthy subjects were also studied as the controls. A significant reduction of serum intact osteocalcin (i-OC) was found in the diabetic groups (p0.01). On the other hand, circulating levels of tartrate resistant acid phosphatase (TRAP) in the diabetic patients were significantly higher than those in the controls (p0.01). Interestingly, a significantly negative relationship was observed between BMD and serum TRAP (p0.01), although no significant relationship was noted between BMD and serum i-OC in diabetic patients. Urinary excretion of SOR was significantly elevated in the diabetic patients when compared with the controls (p0.01). In addition, a significantly positive correlation was observed between serum TRAP and urinary SOR (p0.01), but not between serum i-OC and urinary SOR. Elevated serum TRAP in diabetes was reduced after the administration of aldose reductase inhibitor (p0.05). It seems most likely that the increase in osteoclastic function probably due to accelerated polyol pathway plays a crucial role in the pathogenesis of decreased bone mineral content in male patients with type 2 diabetes.

Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP- d -glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. AB079064 ), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP- d -glucuronic acid to UDP- d -xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.

The bamboo, woody monocot, has two types of parenchyma cells in the ground tissues of its culm, in contrast to a single type of parenchyma cell in rice, maize and other major crop species. The distribution of cell wall components, including lignin, (1-->3), (1-->4)-beta-D-glucans (MGs), the highly-substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans (lbXs) in ground parenchyma tissue of Phyllostachys heterocycla var. pubescens culms was studied at various developmental stages using light microscopy (LM), UV-microscopy, transmission electron microscopy (TEM) and immunolabeling techniques. The short parenchyma cell walls were lignified in 2-month-old bamboo culms just as the long parenchyma cell walls were. The lignified regions were confined to the portions in contact with the long parenchyma cell walls, while the walls at the cell corner region never lignified, even in 7-year-old culms. Significant differences were also found in the hemicellulose distribution between the short and long parenchyma cell walls. In bamboo parenchyma tissue, MGs were localized in short parenchyma cell walls and few were found in long parenchyma cell walls in both young and 7-year-old culms. The distribution of hsGAXs was similar to that of MGs in young culms, but they only appeared in the cell corner region of short parenchyma cells in old culms. Low-branched xylans were distributed in the lignified, but not in unlignified parenchyma cell walls. Based on this evidence, the differences of function in both short and long parenchyma cells in a bamboo culm are discussed.

Polyclonal antibodies which recognized highly substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans and did not cross-react with each other, were raised in order to examine localization of these epitopes in internodes of maize. Immunofluorescent labeling revealed different pattern between two succeeding developmental stages. The hsGAX epitope was localized evenly in primary walls in all tissue types, and strongly in unlignified secondary walls in phloem. However, lignified secondary walls in protoxylem, parenchyma and a part of fibers were faintly labeled with this epitope. Moreover, the epitope showed limited binding in lignified parenchyma and fiber walls at ultrastructural level. Low-branched xylan epitope was localized evenly throughout lignified walls in all tissue types. This epitope was also localized only in lignified walls of other organs such as leaf, root apex and dark-grown mesocotyl. Low-branched xylans are significantly related to lignification. Localization of hsGAX epitope in their organs was similar to that in internodes. The hsGAX epitope was distributed both in unlignified walls of all tissues and in lignified walls of parenchyma and annular thickening of protoxylem. We propose that hsGAX has separate functions in lignified and unlignified tissues. In conclusion, at tissue level, hsGAX is localized mainly in unlignified walls, and low-branched xylans in lignified walls.

In order to investigate the pathogenic role of bone resorption by osteoclasts in altered bone metabolism in non-insulin-dependent diabetes mellitus (NIDDM), the circulating levels of tartrate resistant acid phosphatase (TRACP) were simultaneously determined with osteocalcin, in rat models of NIDDM, i.e., genetic Wistar fatty rats and neonatally streptozotocin-induced diabetic rats (NSZ rats). In Wistar fatty rats exhibiting hyperglycemia and hyperinsulinemia, plasma TRACP was 40.0+/-0.4 U/l (mean+/-SE), significantly higher than that of 32.8+/-1.3 U/l in their lean littermates (p0.01). Bone length, bone strength, and weight of powdered bone in Wistar fatty rats were significantly decreased compared to control rats (p0.02-0.001). On the other hand, plasma TRACP in NSZ rats was 13.6+/-1.0 U/l, significantly lower than that of 31.4+/-1.2 U/l in their controls (p0.01). In addition, there were positive correlations between circulating TRACP and insulin levels in both NIDDM rat models (p0.05-0.01). Furthermore, plasma osteocalcin levels in these NIDDM models were significantly decreased than those of their corresponding controls (p0.001). Consequently, in Wistar fatty rats with hyperinsulinemia, it is suggested that the bone formation by osteoblasts was decreased, while the bone resorption by osteoclasts was increased. In contrast, in NSZ rats with hypoinsulinemia, both of bone formation and resorption were speculated to be decreased, indicating the decreased bone turnover. These results suggest that, although the deterioration in the osteoblastic function can be commonly observed in NIDDM animal models, the osteoclastic function is heterogeneous under NIDDM conditions.

Enzyme-linked immunosorbent assay (ELISA), gel-diffusion precipitin test (GDPT), and indirect haem agglutination test (IHAT) were evaluated for the detection of antibodies to Pasteurella multocida in both naturally and experimentally infected rabbits. A total of 285 rabbit serum samples from 7 rabbit colonies were tested by ELISA, GDPT, and IHAT, and nasal cultures were taken coincidentally to use as the standard in the serological tests. There was better correlation (98.0%) between the results of ELISA and positive nasal culture than between the GDPT (86.3%) or IHAT (23.5%) and positive nasal culture. In addition, ELISA and GDPT were positive in 26 (11.1%) and 21 (9.0%) of 234 serum samples from nasal culture negative rabbits, respectively. In experimentally infected rabbits, antibodies detected by the ELISA and GDPT began to rise one to 3 weeks post-inoculation. IHAT did not detect antibodies. These results are discussed in terms of value to serodiagnosis of rabbit pasteurellosis.

Pradimicins T1 and T2, new members of the pradimicin family of antibiotics, were produced by an actinomycete strain AA3798. Pradimicin T1 exhibited potent activity against a wide spectrum of fungi in vitro and demonstrated efficacy against systemic Candida albicans and Aspergillus fumigatus infections in mice.

BMS-181184 is a new semisynthetic pradimicin derivative with a broad-spectrum antifungal activity. In a search for actinomycetes producing BMS-181184, 4 strains of Actinomadura sp. isolated from soil samples were found to produce the antibiotic under conditions of directed biosynthesis. Among them, Actinomadura sp. AB1236 proved most useful in the production of BMS-181184 when fermented in a medium containing D-serine and D-cycloserine. A minor product isolated from the broth of strain AB1236 was identified as the dexylosyl analog of BMS-181184, which was also obtained by acid hydrolysis of BMS-181184.

It has been observed that repeated and prolonged beta-agonist treatment causes the impairment of beta-adrenergic function, so-called “desensitization” or “down regulation”. To clarify the mechanism of down regulation, the following experiment was performed using guinea pig lungs. Animals were divided into four groups: (1) In the metaproterenol groups, guinea pigs were treated with metaproterenol (10 mg/kg/day) by intraperitoneal injection once a day for 1 day or for 7 successive days (2) In the control groups, guinea pigs were treated with saline by the same procedure as in the metaproterenol groups. In the group treated with metaproterenol for 7 days, there was a 45% reduction in the number of beta-adrenoceptors and a 62% reduction in adenylate cyclase activity, compared with those of the control group. However, there were no significant changes in the dissociation constant (Kd) of the receptors. On the other hand, no reduction in the number of beta-adrenoceptors and adenylate cyclase activity was observed in the group treated with metaproterenol once a day for 1 day, compared with those of the control group. Phospholipase (PLase) activity in the lung microsomes of guinea pigs injected with metaproterenol for 1 day and for 7 days was elevated by 14.4 and 33.1%, respectively, compared with that of the control groups. Phospholipid contents of lung membranes prepared from the animals treated with metaproterenol for 7 days were significantly decreased compared with those of the control group, though in the group treated with metaproterenol once a day for 1 day, phospholipid contents did not differ from those of the control. Lung membranes treated with PLase A2 revealed decreases both in the number of beta-adrenoceptors and adenylate cyclase activity, dose dependently. These results and the fact that membrane phospholipids are involved in the beta-adrenoceptor system suggest that down regulation observed during beta-agonist administration is, at least in part, attributed to degradation of phospholipids of lung membranes by the persistent activation of PLase in the tissue.

DDD mouse embryos at 12–16 days of gestation have a putative ‘fetal’α-globin polypeptide chain, detected by its lower electrophoretic mobility than the authentic α-globin chain on acidic urea-Triton gel. When hepatic erythroid cells of DDD embryos were contaminated with yolk sac erythroid cells, two α-bands were visible, but only ‘fetal’α seemed to be synthesized, because in embryos of strain ddY which share a common ancestry with DDD, only ‘fetal’α was detectable by autoradiography when the hepatic erythroid cells of embryos of more than 15 days of gestation showed an authentic adult α-globin chain other than this ‘fetal’α-globin chain. If this ‘fetal’α-globin chain is structurally different from the yolk sac α and adult peripheral blood α-globin chains, then switching over of the transcription of α-globin polypeptide chain occurs in hepatic erythroid cells of mid-late DDD embryos. However, it is still possible that this ‘fetal’α-globin chain is formed by post-translational modification of the authentic α-globin chain. The α-globin chains of hepatic erythroid cells of C57BL/6 and BALB/c embryos had similar mobilities to those of yolk sac and adult erythroid cells.

The present report deals with the fishery biology of Parapristipoma trilineatum, Pomadasyidae, with particular reference to maturity and spawning habits, on 1242 individuals taken from corstal areas of Kumano-nada (Kii Peninsula, central Japan) from March 1978 to August 1979. The larger half of specimens have a pair of gonads bilaterally asymmetrical in weight. Of these, the specimens with dextral gonads dominate over the specimens with sinistral ones. The development of ovarian egg proceeds uniformly throughout the ovary. The gonad index can effectively be utilized as an indicator representing the state of maturity of ovary. The index of matured female is represented as “GI≥3”. The spawning takes place from June to August. The surface water temperature in the season ranged from 23°C to 28°C. The group maturity rates of female for each age group were estimated as follows: 1-age, 0%; 2-age, 36.4%; 3-age, 86.2%; 4-8-ages, 100%. The biological minimum sizes were estimated at about 160mm for both sexes. The number of ovarian eggs larger than 0.3mm in diameter (E) was given by the following equation: E=1.49L5.00×10-7 (L, fork length in mm). The estimated number of eggs spawned (Es) was obtained by the following equation: Es=5.17L5.10×10-8.

In order to check genetic purity of inbred strains of mice, we surveyed five biochemical variants in certain 69 strains, some of which had established from Japanese fancy mice. The loci examined were Hemoglobin beta-chain, Malic enzyme (supernatant form), Isocitrate dehydrogenase (supernatant form), Serum esterase-1, and Serum esterase-2. These loci were all in homozygous states as far as examined, however some subline divergences were found in a few strains, that is to say, sublines were found to be fixed with different alleles as some loci, Important of biochemical marker genes to check genetic purity of strains and several derived problems are mentioned.

A fetal hemoglobin variant, designated hemoglobin FHouston, was found in the cord blood sample of a healthy, term Negro infant. The variant, comprising about 15 per cent of the total cord blood hemoglobin, diminished concomitantly with hemoglobin F, and it was barely detectable in the blood when the infant was 4 months old. The hemolysates of the parents and two siblings resolved into the usual adult pattern, but a trace amount of a fraction similar to hemoglobin FHouston was present in the father’s hemolysates and not in the mother’s. The ultraviolet absorption spectrum indicates that hemoglobin FHOUSTON contains γ polypeptide chains, and immunologic studies reveal the presence of both α and γ chains. In hybridization tests the alteration appears in the γ chain. Peptide chromatograms of hemoglobin FHouston indicated the presence of α and γ chains, but failed to reveal an abnormality. Amino acid analyses suggest that there may be a substitution of an alanyl for a glutamyl residue.