Ventilatory and behavioural responses of the marbled sole Pseudopleuronectes yokohamae to progressive hypoxia.

This study identified ventilatory and behavioural responses in the marbled sole Pseudopleuronectes yokohamae under experimentally induced progressive decreases in dissolved oxygen (DO) levels. Ventilation frequency showed an increase with decreasing DO levels from normoxia to 2·75 mg O2 l-1 , followed by a decrease in ventilation frequency at decreased DO levels from 2·00 to 0·75 mg O2 l-1 . At DO levels below 2·00 mg l-1 , behaviours at the bottom were suppressed, whereas avoidance behaviours increased. A decrease in avoidance behaviours was observed from 1·00 to 0·75 mg O2 l-1 . Upside-down reversal and incapacitation at DO levels of 1·00-0·75 mg O2 l-1 suggested that sublethal effects on P. yokohamae were induced. The responses observed before the sublethal DO level could be interpreted as an effort to maintain oxygen uptake, reduce routine activities and facilitate avoidance. The observed DO level thresholds that induce behavioural responses, in addition to sublethal effects, indicate hypoxia-tolerance that is important for understanding the effects of hypoxia on coastal ecosystems.

Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation.

RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA scaffold. An electrophoretic mobility shift assay and atomic force microscopy (AFM) analysis revealed that most of the designed circular permutants formed an RNP nano-object. Moreover, the alteration of the enhanced green fluorescent protein (EGFP) orientation was confirmed with AFM by employing EGFP on the L7Ae permutant on the RNA. The results demonstrate that targeted fine-tuning of the stereo-specific fixation of a protein on a protein-binding RNA is feasible by using the circular permutation technique.

Severe Staphylococcus lugdunensis keratitis.

We report a severe case of Staphylococcus lugdunensis (S. lugdunensis) keratitis presenting as suppurative keratitis in a 77-year-old woman. The patient's chief complaint was eye pain and decreased visual acuity in her right eye. Suppurative keratitis with a severe corneal abscess was diagnosed by a slit-lamp ophthalmic examination. The causative organism was identified as S. lugdunensis by bacterial culture, using a corneal abrasion specimen. She was treated with an intravenous drip infusion of ceftazidime and instillation of gentamicin sulfate ophthalmic solution (six times daily) and ofloxacin ophthalmic ointment (once daily before bedtime) as empiric therapy. Her hospital course was complicated by a corneal perforation of her right eye. The antibiotic susceptibility for S. lugdunensis was sensitive, but with a slightly high MIC for antibiotics used in empiric therapy. The therapeutic drug was changed to levofloxacin ophthalmic solution. The corneal abscess left a scar after healing. Representative causative organisms of suppurative keratitis include Pseudomonas aeruginosa and Streptococcus pneumoniae, but care must be taken in cases involving rare causative organisms. Empiric therapy is necessary for rapidly progressing suppurative keratitis, but a detailed examination of the causative organism is important for therapeutic planning before empiric therapy.

Modular engineering of a Group I intron ribozyme.

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4-P6 and P3-P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3-P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3-P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3-P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.

Saponins can cause the agglutination of phospholipid vesicles.

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4-0.5 by a precipitation assay using 14C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl alpha-L-arabinofuranoside but not by p-nitrophenyl beta-D-glucopyranoside or arabinogalactan. The results indicated that the alpha-L-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles.

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.

A purification method for a molecular complex in which a scaffold molecule is fully loaded with heterogeneous molecules.

An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.

A trifunctional, triangular RNA-protein complex.

Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA-protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single triangular RNP via RNA kissing loop (KL) interactions. This RNP-based approach can be used as an alternative tool to produce unique, multifunctional molecules with customized dimensions, functions, and targets.

Revised structure of the peptide lactone antibiotic, TL-119 and/or A-3302-B.

The peptide lactone antibiotic TL-119 and/or A-3302-B was chemically synthesized in order to confirm the proposed structure. The synthetic compound was different from both natural TL-119 and A-3302-B in their physicochemical properties and in biological activity. Re-examination of the configuration of the constituent amino acid residues in natural TL-119 and/or A-3302-B indicated that natural TL-119 and A-3302-B contains D-aThr instead of the original L-Thr. We tentatively propose a revised structure for TL-119 and/or A-3302-B.

3 alpha-hydroxy-oleanane-type triterpene glycosyl esters from leaves of Acanthopanax spinosus.

Three novel 3 alpha-hydroxy-oleanane-type triterpene oligoglycosyl esters, spinoside C1, C4 and C5, were isolated from the leaves of Acanthopanax spinosus. The structures were established to be 28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl esters of 3 alpha, 29-dihydroxy-olean-12-ene-28-oic acid, 3 alpha, 29-dihydroxy-23-oxo-olean-12-ene-28-oic acid and 3 alpha, 23,29-trihydroxy-olean-12-ene-28-oic acid.

Saponins from leaves of Acanthopanax sieboldianus.

Two new triterpenoid saponins named sieboldianoside A and B were isolated from the leaves of Acanthopanax sieboldianus together with five known triterpenoid saponins, kalopanax-saponins A and B, saponin A, CP3, sapindoside B, and a known flavonol glycoside, kaempferol 3-O-rutinoside. On the basis of chemical and spectral evidence, the structures of the new saponins (sieboldianoside A and B) were concluded to be alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->6)- beta-D-glucopyranosyl esters of hederagenin and oleanolic acid 3-O-beta-D- xylopyranosyl(1-->3)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopy ranosides, respectively.

Oleanolic acid saponins from root bark of Aralia elata.

Three new oleanolic acid glycosides, tarasaponins I-III, were isolated as their methyl esters from the root bark of Aralia elata together with four known glycosides, the methyl esters of chikusetsusaponins IVa, IV, 28-desglucosyl-chikusetsusaponin IV and pseudoginsenoside RT1. Tarasaponins I-III were characterized as oleanolic acid 3-O-[beta-D-glucopyranosyl(1-->3)][alpha-L-arabinofuranosyl(1-->4)[- beta-D-glucuronopyranoside, oleanolic acid 3-O-[beta-D-xylopyranosyl(1-->2)][beta-D-galactopyranosyl(1-->3)]-beta- D-glucuronopyranoside and beta-D-glucopyranosyl oleanolate 3-O-beta-D-galactopyranosyl(1-->3)-beta-D-glucuronopyranoside, respectively.

Oleanolic acid saponins from root-bark of Aralia elata.

Five new oleanolic acid glycosides, tarasaponins III-VII, were isolated from the dried root-bark of Aralia elata together with stipuleanoside R2, in addition to eight saponins previously reported. They were characterized as oleanolic acid 3-O-[beta-D-xylopyranosyl(1-->2)] [beta-D-glucopyranosyl(1-->3)]-alpha-L-arabinopyranoside, beta-D-glucopyranosyl oleanolate 3-O-[beta-D-glucopyranosyl(1-->2)][alpha-L-arabinofuranosyl (1-->4)]-beta-D-glucuronopyranoside, beta-D-glucopyranosyl oleanolate 3-O-[beta-D-xylopyranosyl(1-->2)][beta-D-galactopyranosyl (1-->3)]-beta-D-glucuronopyranoside, beta-D-glucopyranosyl oleanolate 3-O-[beta-D-xylopyranosyl(1-->2)][beta-D-glucopyranosyl (1-->3)-alpha-L-arabinopyranoside, respectively.

Steroidal glycosides from the subterranean parts of Liriope spicata var. prolifera.

In a continuation of phytochemical studies on the underground organs of Liriope spicata var. prolifera, four new steroidal glycosides, lirioproliosides A-D, along with two known compounds, 25(S)-ruscogenin 1-O-[alpha-L-rhamnopyranosyl(1-->2)[beta-D-xylopyranosyl (1-->3)]-beta-D-fucopyranoside and ophiopogonin A, were identified. The structures of lirioproliosides A-D were established by a combination of spectroscopic and chemical methods as 25(S)-ruscogenin 1-O-[alpha-L-rhamnopyranosyl(1-->2)][beta-D-xylopyranosyl (1-->3)]-beta-D-fucopyranoside-3-O-alpha-L-rhamnopyranoside, 25(S)-ruscogenin 1-O-[3-O-acetyl-alpha-L-rhamnopyranosyl(1-->2)]-beta-D-fucopyranoside, 25(S)-ruscogenin (1-O-[2-O-acetyl-alpha-L-rhamnopyranosyl (1-->2)]-beta-D-fucopyranoside and ruscogenin (1-O-[2-O-acetyl-alpha-L-rhamnopyranosyl(1-->2)]-beta-D-fucopyranoside, respectively. Among these steroidal glycosides, ophiopogonin A and lirioprolioside B, and lirioproliosides C and D, were isolated as epimeric pairs.

Early intervention for very-low-birth-weight infants.

To assess the efficacy of early intervention (EI) for very-low-birth-weight (VLBW) infants, we evaluated 62 2 year old children who were enrolled in an EI program and 48 control subjects aged 2 years. We determined the subjects' developmental quotients (DQ) and obtained information about the parents' evaluation of the children from a questionnaire sent to the parents. There was no significant difference in the DQ between the EI group and the control group. However, based on the responses to the questionnaire, subjects in the EI group showed slight, but statistically marginally significant, improvements in behavioral problems, especially a decrease in hyperkinesia, in adjusting to a circadian sleep cycle, and an improvement in language development, as compared with the control group (P < 0.1). Thus, EI for VLBW infants is considered useful to enhance some areas of development.

Chemical characterization of new antibiotics, cerexins A and B. (Studies on antibiotics from the genus Bacillus. II)

Acid hydrolysis revealed that the antibiotic cerexin A is constructed with aspartic acid (3), threonine (1), serine (1), valine (2), allo-isoleucine (1), gamma-hydroxylysine (1), tryptophan (1), and a variety of fatty acid residues. The essential difference between cerexins A and B is concluded to be replacement of serine and one valine residue in cerexin A by glycine and phenylalanine in cerexin B. Isolation of a new amino acid L-threo-gamma-hydroxylysine is also described.

The structure of brevistin. Studies on antibiotics from the genus Bacillus. X.

Acid hydrolysis revealed the constituent amino acids by brevistin as follows: aspartic acid (D-form 2, L-form 1), L-threonine (1), glycine (1), sum of L-valine and L-isoleucine (1), L-phenylalanine (1), L-trytophan (1), and 2,4-diaminobutyric acid (D-form 1, L-form 1). The constituent fatty acid was elucidated to be anteisononanoic acid by gas chromatography-mass spectrometry. A lactone linkage was proved between phenylalanine and the hydroxy group of threonine. Opening the lactone ring with dilute alkali afforded brevistinic acid. Deacylation with an enzyme preparation, Polymyxin Acylase, gave deacyl brevistinic acid. Cleavage reaction with N-bromosuccinimide, sequential analysis by EDMAN degradation and some additional evidences clarified the total structure of brevistin.

The structure of cerexin B (studies on antibiotics from the genus Bacillus. XVII).

The structure of cerexin B was examined. The constitutent fatty acids were elucidated by gas chromatography and mass spectrometry to be beta-hydroxy isodecanoic acid, beta-hydroxy decanoic acid, beta-hydroxy isoundecanoic acid and beta-hydroxy anteisoundecanoic acid. The configurations of constituent amino acids were determined as asparagine (2D, 1L), valine (D), phenylalanine (D), allo-threonine (D), tryptophan (D), and allo-isoleucine (D) from their optical activities. Treatment with conc. hydrochloric acid cleaved at the gamma-hydroxylysine residue to give two peptide fragments, one of which (the N-terminal side) was then deacylated with Polymyxin Acylase. Their amino acid sequences were examined by EDMAN degradation. From the results and analogy to cerexin A, the structure of cerexin B was deduced.

The amino acid sequence of cerexin A (studies on antibiotics from the genus Bacillus. VII.

N-Bromosuccinimide cleavage reaction on cerexin A liberated allo-isoleucine. Treatment with conc. hydrochloric acid cleaved the antibiotic into two peptide fragments selectively at gamma-hydroxylysine residue. Deacylation with an enzyme preparation from Pseudomonas sp. afforded deacyl cerexin A. The amino acid sequences of these peptide fragments were examined by Edman degradation. From all the results, the entire amino acid sequence of cerexin A was deduced.

Structures of agglomerins.

Structures of a series of new antibiotics, agglomerins A, B, C and D, which are active against a variety of anaerobic bacteria, were determined to be 1-acyl-2,3-dihydroxy-1,3-butadiene-1-carboxylic acid, (1----3)-gamma-lactones, i.e., 2-acyl-4-ylidenetetronic acids with different hydrocarbon chains in the acyl group. Their common chromophore exhibited tautomerism in solution. The relationship of their structure to the activity against anaerobes is discussed.