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.

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.

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.

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.

Artificial modules for enhancing rate constants of a Group I intron ribozyme without a P4-P6 core element.

In this paper we report newly selected artificial modules that enhance the kcat values comparable with or higher than those of the wild-type ribozyme with broad substrate specificity. The elements required for the catalysis of Group I intron ribozymes are concentrated in the P3-P7 domain of their core region, which consists of two conserved helical domains, P4-P6 and P3-P7. Previously, we reported the in vitro selection of artificial modules residing at the peripheral region of a mutant Group I ribozyme lacking P4-P6. We found that derivatives of the ribozyme containing the modules performed the reversal of the first step of the self-splicing reaction efficiently by using their affinity to the substrate RNA, although their kcat values and substrate specificity were uninfluenced and limited, respectively. The results show that it is possible to add a variety of new domains at the peripheral region that play a role comparable with that of the conserved P4-P6 domain.

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.

[Histological study of the effect of conjunctival transplantation on rabbit cornea with alkali burned].

PURPOSE: A histopathological study to investigate the efficacy of conjunctival auto-transplantation on alkaline chemical burns of the ocular surface. MATERIAL AND METHODS: An alkaline chemical burn model was developed in one eye of rabbits using 0.1 N NaOH solution. In one group conjunctival transplantation was performed. A control group did not receive conjunctival transplantation. A histological follow-up study was performed by light microscopy with hematoxylin eosin stain and periodic acid Schiff staining by fluoro-microscopy using epithelial Keratin-AE 5 antibody, and by transmission electron microscopy(TEM). RESULTS: The transplanted group showed scar formation between the transplanted conjunctival tissue and the sclera. At 20 days and 8 weeks after the transplantation, neovascularization and cell infiltration in the subepithelium of the limbus was decreased compared with the transplanted group at 10 days. The control group showed a decrease of cell infiltration in the limbal area compared with the group at 10 days, but conjunctival tissue with thick collagenous tissue and neovascularization instead of scar tissue developed on the injured cornea. AE 5 positive cells were not found in the limbus in either group. In the transplanted group, in TEM, the basement membrane of transplanted conjunctiva showed less irregularity, and degenerated fibroblasts were present at the margin of the scar tissue. In the control group, the basement membrane of the conjunctiva showed an irregular pattern and fibroblasts beneath the conjunctival epithelium had large nuclei. CONCLUSION: In the transplanted group, scar tissue developed and suppressed cell infiltration, neovascularization, and conjunctival tissue in the injured cornea and secured re-structuring of the limbal tissue.

Immunosuppressive effect of cholera toxin B on allergic conjunctivitis model in guinea pig.

PURPOSE: To investigate the new method of immunotherapy using cholera toxin B (CTB) in experimental allergic conjunctivitis. METHODS: We used 21 white Hartley guinea pigs. The animals were sensitized by intraperitoneal injection of ovalbumin (100 microg/mL) and albumin hydroxide (5 mg/mL) repeated after an interval of 2 weeks. One week after the second injection, conjunctivitis was induced by topical instillation of ovalbumin (5 mg/mL). The animals were divided into two groups, CTB group and control group. The CTB group underwent pretreatment of topical instillation of CTB (4 microg/30 mL) and ovalbumin (10 microg/30 mL), three times a day for 3 days, 1 week before the intraperitoneal injection. The control group did not undergo the pretreatment. Clinical examination was performed at 0.5, 6, and 24 hours after the development of conjunctivitis. Histological examination was performed at 6 and 24 hours. RESULTS: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation of ovalbumin. The allergic reaction score was significantly less in the CTB group than in the control group (Mann-Whitney U-test: P <.01). The clinical reactions subsided after 6 hours. The CTB group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival epithelium, than the control group at 6 and 24 hours. CONCLUSION: Pretreatment with topical CTB and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis.

Immunosuppressive effect of cholera toxin B on allergic conjunctivitis model in the guinea Pig

Purpose: To investigate the immunosuppressive effects of mucosal immune therapy in experimental allergic conjunctivitis.Method: We used 11 white Hartrey guinea pigs divided into two groups. Six animals (treated group) received pretreatment with topical instillation of cholera toxin B (4 &mgr;g/30 ml) and ovalbumin (10 &mgr;g/30 ml). The other group of 5 animals served as control. All the animals received intra-abdominal injection of ovalbumin (100 g/mL) and aluminum hydroxide (5 mg/mL) repeated twice 2 weeks apart. Allergic conjunctivitis was induced by topical instillation of ovalbumin solution (5 mg/mL) 1 week after the above procedure. Result: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation. The allergic reaction was significantly less in score in the treated than in the control group (Mann-Whitney U-test: P <.01). The clinical findings subsided after 6 hours. The treated group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival epithelium, than in the control group at 6 and 24 hours.Conclusion: Pretreatment with topical cholera toxin B and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis.

[Immunohistochemical study of interstitial keratitis in an animal model].

PURPOSE: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA). METHODS: A mixture of OA (5 micrograms/ml) and Freund's complete adjuvant was initially injected subcutaneously (0.4 ml) into the rabbits' back and footpad (0.2 ml). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 ml) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 ml) was injected into the corneal stroma. We observed the cornea on days 1-3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done. RESULTS: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils in to the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10. CONCLUSIONS: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

[Immunosuppressive effect of cholera toxin B on allergic conjunctivitis model in the guinea pig].

PURPOSE: To investigate the immunosuppressive effects of mucosal immune therapy in experimental allergic conjunctivitis. METHOD: We used 11 white Hartrey guinea pigs divided into two groups. Six animals (treated group) received pretreatment with topical instillation of cholera toxin B (4 micrograms/30 ml) and ovalbumin (10 micrograms/30 ml). The other group of 5 animals served as control. All the animals received intraabdominal injection of ovalbumin (100 g/ml) and aluminium hydroxide (5 mg/ml) repeated twice 2 weeks apart. Allergic conjunctivitis was induced by topical instillation of ovalbumin solution (5 mg/ml) one week after the above procedure. RESULT: Both groups developed palpebral and bulbar edema with hyperemia 30 minutes after instillation. The allergic reaction was significantly less in score in the treated than in control group (Mann Whitney U-test: p < 0.01). The clinical findings subsiced after 6 hours. The treated group showed less eosinophilic infiltration in the conjunctiva and the limbus, particularly in the conjunctival and the limbus, particularly in the conjunctival epithelium, than in control group at 6 and 24 hours. CONCLUSION: Pretreatment with topical cholera toxin B and antigen suppresses clinical and histological findings in experimentally induced allergic conjunctivitis.

Triterpenoid saponins of Acanthopanax nipponicus leaves.

Five new triterpenoid saponins, nipponosides A-E (1, 3-6), were isolated from Acanthopanax nipponicus leaves, along with a known saponin, kalopanaxsaponin G (2). Nipponosides A-E were characterized as the 28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->6)-beta -D-glucopyranosyl ester of 3-oxohederagenin, oleanolic acid 3-O-beta-D-glucopyranoside, gypsogenin 3-O-beta-D-glucopyranoside, 3beta,23,29-trihydroxyolean-12-en-28-oic acid, and 3beta,20alpha, 23-trihydroxy-30-nor-olean-12-en-28-oic acid, respectively. The structures of these new compounds were based on chemical and spectral methods.

[A case of corneal abscess with impetigo contagiosa].

Impetigo contagiosa often occurs on the face of children. Although the eyelid and conjunctiva are frequently affected, no corneal involvement has been reported in Japan. We encountered a case of impetigo contagiosa with corneal abscess. The patient, an 8-year-old boy, complained of pain and presented with corneal abscess and iritis in the right eye as well as eruptions in the inferior eyelid and around the anterior nostrils. He was diagnosed as having impetigo contagiosa with corneal abscess. Because the corneal abscess and eruptions occurred at the same time and coagulase-positive Staphylococcus with identical drug sensitivity was isolated from both lesions, we considered the corneal lesion as a part of the impetigo contagiosa. When impetigo contagiosa occurs around the eye, especially on the eyelids, a careful examination of the anterior segment of the eye is needed.

[Clinical features of 10 cases with phlyctenular keratitis].

We investigated 15 eyes of 10 patients with phlyctenular keratitis. The patients were 6 men and 4 women, and their ages ranged between 3 and 56 years (mean, 17.8 years). The follow-up periods ranged from 0.5 to 48 months (mean, 14 months). Clinically, complications in many cases were papillary hyperplasia in palpebral conjunctiva, meibomian gland infarct, chalazion, and blepharitis. Microbial examination from the lid margin before treatment revealed bacteria in 4 eyes of 4 patients out of 9 eyes of 8 patients. The level of serum specific IgE antibody was high in only 1 patient out of 6. We classified the clinical course into 3 stages, namely, infiltrative, nodule, and cicatricial stages. We also investigated the transition of the disease among the stages. All the cases of the infiltrative stage progressed to the nodule stage, and 92% of the nodule stage went on to the cicatricial stage. However 14% of the cicatricial stage cases reverted to the infiltrative stage and 7% to the nodule stage.

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.

Immunohistochemical study on follicular dendritic cell of conjunctiva-associated lymphoid tissue.

We performed an immunohistochemical study of follicular dendritic cells (FDC) in the follicular area of the conjunctiva-associated lymphoid tissue (CALT). Hartley guinea pigs were sensitized with a topical application of an emulsion of ovalbumin and Freund's complete adjuvant in the eye. They were divided into four groups. The control (group A) underwent no sensitization. The sensitized animals were studied at 1 week (group B1) or 2 weeks (group B2) after the sensitization. Additional sensitization at 1 week after the initial sensitization was also performed (group C). Histological methods included methylgreen pyronine staining, alpha-naphthylacetate esterase staining, and enzyme-antibody method against S-100 protein. The uptake of topically applied peroxidase-anti-peroxidase (PAP) in CALT was also examined histologically. In each group, positive reticular patterns by alphanaphthyl acetate esterase staining and immunoperoxidase staining with anti-S-100 protein antiserum were found in the CALT follicular area. The positively stained cells were found to be dendrite cells by immunoelectronmicroscopy. An uptake of PAP was found in the CALT follicular area, suggesting the function of trapping and retaining antigen-antibody complex by FDC. It was concluded that dendritic cells in the CALT follicular area were identified to be FDC.

[Histological examination of lymphoid follicles in conjunctiva-associated lymphoid tissue in guinea pigs--Report 1. Histological characteristics of follicular dendritic cells].

We histologically examined the existence of follicular dendritic cells (FDCs) in the follicular area of conjunctiva-associated lymphoid tissue (CALT). Animals used in the experiment were Hartley guinea pigs sensitized with a topical application to the eyes of an emulsion of a mixture of ovalbumin and Freund's complete adjuvant in equal amounts. The animals were divided into 4 groups: a group with no eye drops administered (Group A), a group examined 1 week after administration of eye-drops (Group B-1), a group examined 2 weeks after administration (Group B-2), and a group given booster administration of eye-drops (Group C). These animals were examined by methyl green pyronine staining, alpha-naphthylacetate esterase staining, and enzyme-antibody methods against S-100 protein or major histocompatibility complex class II (MHC class II) antigen. Also peroxidase-anti-peroxidase (PAP) was applied to the eyes and the uptake of PAP in CALT was observed histologically. In each group, positive reticular stains to alpha-naphthyl-acetate esterase staining and S-100 protein were found in the CALT follicular area. The positive cells were found to be dendrite cells by immunoelectron microscopy. An uptake of PAP was seen in the CALT follicular area, suggesting the function of trapping and holding the antigen-antibody complex by FDCs. It was concluded that dendritic cells present in the follicular area of CALT were FDCs.

[Isolation and structure elucidation of cirensenosides Q and R].

Two new saponins (cirensenoside Q and R) were isolated from the leaves of Oplopanax elatus. Their structures were elucidated as follows: Q, gypsogenin 3-O-beta-glucopyranosyl-28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D- glucopyranosyl(1-->6)-beta-D-glucopyranoside; R, 3 beta-hydroxylup-20(29)-ene-23-al-28-oic acid 3-O-beta-D-glucopyranosyl-28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D- glucopyranosyl(1-->6)-beta-D-glucopyranoside, 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.