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Rhodococcus rubropertinctus N82 possesses unique regiospecific hydroxylation activity in biotransformation of compounds. In this study, the ability of whole cells of the strain R. rubropertinctus N82 in biotransformation was studied. The hydroxylation activity resulted in transforming 6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LS1) into 2-hydroxy-6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LP1), a pharmaceutical intermediate. By optimizing conditions for the hydroxylating biotransformation using whole cells of R. rubropertinctus N82 as biocatalyst, 3.3 mM LP1 was successfully produced from 4 mM LS1 with a molar yield of 83%. Thus, effective method was newly developed to produce LP1, which is a synthetic intermediate of a platelet inhibitor active pharmaceutical ingredient drug, prasugrel.

The effects of overexpression of Rubisco activase on photosynthesis were studied in transgenic rice expressing barley or maize Rubisco activase. Immunoblot and SDS-PAGE analyses showed that transgenic lines from both gene constructs expressed the foreign Rubisco activase at high levels. The activation state of Rubisco in transgenic lines was slightly higher than that in non-transgenic plants (NT). In addition, light activation of Rubisco was significantly more rapid in transgenic lines compared with NT. These findings indicate that the overexpression of Rubisco activase can enhance Rubisco activation. However, despite enhanced activation of Rubisco in these transgenic plants, the CO(2) assimilation rate at ambient CO(2) conditions was decreased. This decrease in CO(2) assimilation rate was observed in both young developing and mature leaves independent of nitrogen nutrition. The contents of nitrogen and Chl did not differ significantly between transformants and NT; however, Rubisco content was substantially decreased in transgenic lines. There was no evidence for reduced transcription of RbcS or RbcL in these transgenic lines; in fact, transcript levels were marginally increased compared with NT. These results indicate that the overexpression of Rubisco activase leads to a decrease in Rubisco content, possibly due to post-transcriptional mechanisms.

Background: The etiology of inflammatory bowel disease (IBD) is associated with an altered microflora due to a failure of the immune system. This study investigated the expression of the intestinal antimicrobial peptide alpha-defensin, which plays a pivotal role in the regulation of the intestinal microflora in a representative model of IBD, interleukin (IL)-10-deficient mice., Methods: The expression of alpha-defensin/cryptdins in IL-10-deficient mice was assessed by real-time polymerase chain reaction (PCR) and acid/urea polyacrylamide gel (AU-PAGE). The alteration of alpha-defensin/cryptdins expression was compared with the inflammatory grade of mice intestine at various weeks from birth., Results: The weight, length, and inflammation grade of the mouse intestines were assessed at 5, 7, 9, 11, 13, and 15 weeks from birth. While the weight of the large intestine was heavier at 15 weeks after birth in the IL-10-deficient mice than in the control mice, histological inflammation began from 7 weeks after birth. Real-time PCR and AU-PAGE identified a significant decrease in the expression of alpha-defensin/cryptdins at 7 weeks after birth in the IL-10 knockout mice, thus illustrating the involvement of alpha-defensin/cryptdins in the etiology of the intestinal inflammation in IBD. This study also identified the expression of alpha-defensin/cryptdins to be inversely proportional to age until 11 weeks, suggesting a relationship between the formation of the intestinal microflora and a reduction in the expression of alpha-defensin/cryptdins., Conclusions: The altered expression of antimicrobial peptide alpha-defensin may cause the onset of intestinal inflammation due to a failure to regulate intestinal microflora.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] has previously been identified from the lepidopteran larval regurgitant as an elicitor of plant volatile emission. The efficient incorporation of free oxygen into volicitin by Spodoptera litura larvae is demonstrated here by rearing them under (18)O(2) for three days. (18)O-labeling of the hydroxyl group of volicitin was confirmed by liquid chromatography/mass spectrometry-ion trap-time-of-flight (LC/MS-IT-TOF) and suggests the activity of a monooxygenase in volicitin biosynthesis.

Aims: To evaluate whether plasma high molecular weight (HMW) adiponectin provides prognostic information in addition to that obtained from clinical, haemodynamic, and biochemical variables previously known to be associated with a high mortality in chronic heart failure (CHF) patients., Methods and Results: We measured the plasma levels of total and HMW adiponectin, atrial natriuretic peptide, brain natriuretic peptide (BNP), and N-terminal-proBNP (NT-proBNP), and haemodynamic parameters in 449 consecutive CHF patients. Based on body mass index (BMI), patients were classified into three groups: low (<21 kg/m(2), n = 133), normal (21-25 kg/m(2), n = 205), and high (>25 kg/m(2), n = 111). After adjustment for clinical variables associated with CHF including haemodynamics, plasma total adiponectin level was an independent prognostic predictor but HMW adiponectin was not in the overall patient group. On subgroup analyses, in patients with abnormal BMI, plasma total adiponectin level was not an independent prognostic predictor, but in patients with normal BMI, plasma levels of log NT-proBNP (P = 0.017) and log total adiponectin (P = 0.003) were independent prognostic predictors., Conclusion: These findings indicate that total adiponectin is more useful for assessing mortality risk than HMW adiponectin and a high plasma total adiponectin is an independent prognostic predictor especially in CHF patients with normal BMI.

We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine kappa-casein. The amino acid sequence of this peptide is Phe-Leu-Pro-Tyr-Pro-Tyr (FLPYPY), corresponding to peptidic sequence 76-81 of bovine kappa-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10(-9) M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76-80 and 76-82 of bovine kappa-casein respectively, were ineffective.

Preliminary crystallographic studies of a Bowman-Birk type protease inhibitor, AB-I, from adzuki beans (Phaseolus angularis) 'Takara,' and its complex with trypsin were carried out. AB-I, MW 9100 with 82 amino acid residues, crystallizes in a trigonal space group, P3121 (or P3221), with the following unit cell dimensions: a = 68.7, c = 99.7 A. The asymmetric unit contains two dimer molecules. Structure analysis at 5 A resolution revealed the rough appearance of the dimer. The complex between AB-I and trypsin also could be crystallized in a tetragonal space group, P41212 (or P43212), with the unit cell dimensions, a = 55.4 and c = 181.5 A, and Z = 8. The crystallinity seems to be much better than that of the crystals of the inhibitor alone. The other type of inhibitor from adzuki bean, AB-IIa, was also crystallized.

Double-headed protease inhibitors I, IIa, and IIc (AB I, AB IIa, and AB IIc) have been purified from azuki beans "Takara" (Vigna angularis) by conventional chromatographic methods and their amino acid sequences have been determined. AB I, AB IIa, and AB IIc had molecular weights of 9,166, 8,661, and 8,756 daltons, consisting of 82, 78, 79 amino acid residues, respectively. The molecular weights of these inhibitors, determined by gel filtration at pH 8.0, were 18,000 for AB I and 17,000 for both AB IIa and AB IIc, indicating that the inhibitors are dimers. The inhibitors had isoelectric points of 4.7 (AB I), 6.8 (AB IIa), and 6.2 (AB IIc). AB I stoichiometrically inhibited both trypsin and chymotrypsin at a molar ratio of 1 : 1. On the other hand, AB IIa and AB IIc both inhibited trypsin at a molar ratio of about 1 : 2 and also inhibited chymotrypsin, though only weakly. Sequence comparison with other double-headed inhibitors indicated the reactive sites of AB IIa and AB IIc for trypsin to be Lys26-Ser27 and Arg53-Ser54, and those of AB I for trypsin and chymotrypsin to be Lys26-Ser27 and Tyr53-Ser54, respectively. The differences between AB IIa and AB IIc were that AB IIa lacked the C-terminal aspartic acid residue, and that Glu10 and Arg60 in AB IIa were replaced by Gln10 and His60 in AB IIc. A comparison between AB IIa and AB I revealed 25 variant amino acids among the 78 residues of AB IIa; further, Ab IIa lacked 4 amino acid residues in the C-terminal region of AB I.

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.