Structures of azaspiracid analogs, azaspiracid-4 and azaspiracid-5, causative toxins of azaspiracid poisoning in Europe.

Two new analogs of azaspiracid, azaspiracid-4 and azaspiracid-5, isolated from the mussel Mytilus edulis, involved in a newly emerged shellfish poisoning in Europe were determined to be 3-hydroxy-22-demethylazaspiracid and 23-hydroxy-22-demethylazaspiracid, respectively.

Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland.

A new type of food poisoning resulting from ingestion of mussels produced in Ireland occurred in the Netherlands in 1995 and then reoccurred in Ireland in 1997. As the causative agent, azaspiracid, was isolated in pure form and revealed to have a structure entirely unlike other known algal toxins, in vivo studies with mice were carried out to elucidate the pathological injuries caused by the toxin. By per os administration, the toxin caused necrosis in the lamina propria of the small intestine and in lymphoid tissues such as thymus, spleen and the Peyer's patches. Both T and B lymphocytes were injured. Additionally a fatty change was observed in the liver. These injuries distinctly differed from those caused by the representative diarrhetic shellfish toxin, okadaic acid.

Signaling regulation for synergistic effects of substance P and insulin-like growth factor-1 or epidermal growth factor on corneal epithelial migration.

PURPOSE: In a previous report we showed that substance P (SP) and insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) synergistically stimulate corneal epithelial migration. In this study, we used an organ culture system of rabbit cornea to identify which signal transduction system affects corneal epithelial migration. METHODS: Rabbit corneal blocks were cultured in TC-199 culture medium containing various reagents for 24 hours. After the end of cultivation, the length of the path of epithelial migration was measured. RESULTS: Acting alone, protein kinase C (PKC) inhibitors, calphostin C and H-7, each reduced the length of epithelial migration. Tyrosine kinase (TK) inhibitors, genistein and herbimycin A, also acted individually to inhibit epithelial migration. The synergistic stimulatory effects of SP and IGF-1 on corneal epithelial migration were eliminated when PKC inhibitors or TK inhibitors were added. The synergistic effect of SP and EGF was eliminated by TK inhibitors, but only partly suppressed by PKC inhibitors. CONCLUSIONS: These results suggest that the synergistic effect of SP and EGF might require a TK pathway, and that the synergistic effect of SP and IGF-1 might require both PKC and TK pathways.

Differential tyrosine phosphorylation of paxillin in human corneal epithelial cells on extracellular matrix proteins

Purpose: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion.Methods: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and Western blotting to determine tyrosine phosphorylation of paxillin.Results: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix. Conclusion: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.

[Differential tyrosine phosphorylation of paxillin in human corneal epithelial cells on extracellular matrix proteins].

PURPOSE: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion. METHODS: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and western blotting to determine tyrosine phosphorylation of paxillin. RESULTS: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix. CONCLUSION: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.

A sensitive and specific determination method for azaspiracids by liquid chromatography mass spectrometry.

A liquid chromatography/mass spectrometry (LC/MS) method was developed for the sensitive and specific determination of azaspiracid and its two analogs, the causative toxins of azaspiracid poisoning that occurred in the Netherlands and Ireland. The LC/MS method provided a detection limit of 50 pg for azaspiracid. The sensitivity was approximately 8 x 10(4) times greater than the mouse bioassay. The method was used to confirm the presence of azaspiracids in toxic mussels collected at Arranmore Island, Ireland in 1997.

Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland.

Two new analogs of azaspiracid, azaspiracid-2 and azaspiracid-3, were isolated from mussels collected at Arranmore Island, Ireland in 1997 as additional causes of human intoxication. Their structures were determined to be 8-methylazaspiracid and 22-demethylazaspiracid, respectively by NMR and negative ion FAB CID MS/MS experiments.

[Effects of an antidiarrhoeica containing an extract from geranium herb on astringent action and short-circuit current across jejunal mucosa].

A consists of berberine chloride and an extract from geranium herb. To clarify mechanisms of the antidiarrheal effect of Phelloberin-A, we investigated the astringent action by determining its binding activity to rabbit hemoglobin and effects on active transport, which was indicated by short-circuit current (Isc), in rat jejunum by the Ussing chamber technique. The effects of berberine chloride and geranium herb on both the binding activity to hemoglobin and the electrophysiological parameters such as Isc were compared with those of the antidiarrhoeicas, tannic acid, albumin tannate and bismuth subnitrate. Geranium herb, tannic acid and bismuth subnitrate increased significantly the binding activity to hemoglobin at concentrations of > 1 mg/ml, > 0.3 mg/ml and 10 mg/ml, respectively, but berberine or albumin tannate did not. Geranium herb and tannic acid dose-dependently and moderately increased Isc in rat jejunal mucosa and the increase became significant at a concentration of 10 mg/ml. Neither berberine chloride, albumin tannate nor bismuth subnitrate affected Isc. In contrast, cholera toxin, which increases the secretion from intestinal mucosa to the lumen and induces diarrhea, decreased Isc at a concentration of 0.1 mg/ml. The decrease of Isc induced by cholera toxin was antagonized by pretreatment with geranium herb (10 mg/ml), indicating that geranium herb inhibited the toxin-induced increase in secretion. These results suggest that geranium herb possesses an astringent action and moderately increases Isc across the intestinal mucosa. Therefore, the effects may support an antidiarrheal effect of both geranium herb and Phelloberin-A.

Synergistic effect of substance P with epidermal growth factor on epithelial migration in rabbit cornea.

In order to investigate the role of neural regulation in corneal epithelial healing, we examined the effect of substance P (SP) on corneal epithelial migration using an organ culture system of rabbit corneas. We investigated the synergistic effects of SP with (1) growth factors: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta(TGF-beta); (2) extracellular matrix proteins: fibronectin, vitronectin, laminin, and collagen type IV; and (3) cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, and interleukin-6 (IL-6). Rabbit corneal blocks were cultured in the absence or presence of various reagents for 24 hr. The corneal blocks were then fixed, dehydrated, embedded in paraffin and stained by hematoxylin-eosin, and the length of the path of epithelial migration was measured. The addition of SP alone, at concentrations up to 50 microg ml-1, did not affect epithelial migration. EGF, fibronectin, vitronectin, collagen type IV, and IL-6 stimulated epithelial migration, but bFGF, TGF-beta, laminin, IL-1alpha, and IL-1betadid not. The stimulatory effect of EGF on the epithelial migration was enhanced by the presence of SP. This synergistic effect of SP and EGF on corneal epithelial migration was abolished by the addition of an SP antagonist or enkephalinase. Other neurotransmitters (vasoactive intestinal peptide, calcitonin gene-related peptide, acetylcholine chloride, norepinephrine, serotonin) and tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin) were examined, but none exhibited a synergistic effect with EGF. Interestingly, EGF alone stimulated the incorporation of 3H-thymidine into corneal epithelial cells, but the addition of SP with EGF did not enhance this effect. These results demonstrate that SP enhanced the EGF stimulation of corneal epithelial migration in vitro in a specific manner, suggesting a possible role of SP as a modulator of epithelial wound healing.

Combined effects of substance P and insulin-like growth factor-1 on corneal epithelial wound closure of rabbit in vivo.

PURPOSE: We investigated the combined effects of substance P (SP) and insulin-like growth factor-1 (IGF-1) on corneal epithelial wound closure in vivo. METHODS: The corneal epithelium of 16 rabbits was debrided by n-heptyl alcohol treatment; SP (1 mg/ml) and/or IGF-1 (1 microgram/ml) in phosphate-buffered saline (PBS) were administered immediately and at 2, 4, 6, 8, 10, 24, 26, 28, 30, 32, and 34 hours after debridement. Controls received PBS alone. The eyes were stained with fluorescein and photographed at baseline and at 6, 12, 18, 24, 30, 36, and 48 hours after debridement. The wound radii of the epithelial defects were recorded from hour 12 to hour 30, and the rate of healing was calculated by linear regression analysis. RESULTS: The mean (+/-SD) healing rate in the control group was 57.03 +/- 7.88 microns/h. The administration of SP or IGF-1 alone did not affect the healing rates, which were 55.49 +/- 2.49 and 55.57 +/- 7.14 microns/hr, respectively. However, when SP and IGF-1 were combined, the mean healing rate was significantly higher (75.16 +/- 6.68 microns/hr) than that of the control, SP-treated, or IGF-1-treated groups (p < 0.05). CONCLUSIONS: These results demonstrated that SP and IGF-1 synergistically affect corneal epithelial wound closure.

The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin-like growth factor-1.

1. We have previously shown that substance P (SP) and insulin-like growth factor-1 (IGF-1) act synergistically to enhance the migration of rabbit corneal epithelial cells in an organ culture model. The present study was designed to identify the epithelial cell SP receptor that participates in this synergistic effect. 2. Rabbit corneal blocks were incubated for 24 h, then the length of the path of epithelial migration was measured. Reagents tried in the TC-199 culture medium, in the presence or absence of IGF-1, were: SP, agonists of tachykinin receptors NK1, NK2 or NK3 and antagonists of tachykinin receptors NK1 or NK2. 3. The binding characteristics of SP receptors were examined in rabbit cultured corneal epithelial cells by binding assays with [125I]-SP in the presence or absence of excess unlabelled SP or ligands of NK1, NK2 or NK3 receptors. 4. As was demonstrated previously, SP and IGF-1 stimulated epithelial migration when they were added to the culture medium together, but individually they had no effect. NK1 agonists had the same synergistic effect with IGF-1 as did SP, but the NK2 and NK3 agonists did not. Furthermore, the NK1 antagonist abolished the synergistic effect of SP and IGF-1, but the NK2 antagonist had no effect. 5. SP bound specifically to rabbit cultured corneal epithelial cells. The binding affinity was 0.44 nM and there were 2.43 x 10(4) binding sites per cell. The NK1 ligand competed, in a dose-dependent fashion, with the binding of SP to corneal epithelial cells, but neither the NK2 nor NK3 ligand affected binding. 6. We conclude that the SP receptor in rabbit corneal epithelial cells is NK1 and that this receptor participates in the synergistic enhancement of corneal epithelial migration by SP and IGF-1. The precise mechanism(s) of this interaction requires more study. These findings imply that both neural and humoral factors are essential for the maintenance and healing of corneal epithelium.

[Neurotrophic keratopathy--studies on substance P and the clinical significance of corneal sensation].

Neurotrophic keratopathy, which often follows damage to the trigeminal nerve, is clinically characterized by various types of epithelial disorders and melting of corneal stroma. To understand both the pathology of neurotrophic keratopathy and the physiological significance of corneal sensation, we investigated both the cellular and molecular functions of a sensory neurotransmitter, substance P, in corneal epithelial cells. Our findings prompted us to try a new mode of treatment for neurotrophic keratopathy. Substance P, a member of the tachykinin family, is an 11-amino-acid peptide. In an organ culture system using rabbit corneas, substance P alone had no effect on corneal epithelial migration. In the presence of insulin-like growth factor-1 (IGF-1), however, substance P synergistically facilitated corneal epithelial migration in proportion to the concentration of substance P or of IGF-1. Other neurotransmitters (acetylcholine, norepinephrine, serotonin etc.) or tachykinins (neurokinin A, eledoisin etc.) did not show this synergistic effect with IGF-1. Among receptors for the tachykinin family (NK-1, NK-2, or NK-3) only the NK-1 receptor system was involved in the synergistic effect of substance P and IGF-1 on corneal epithelial migration. IGF-1 affected neither the binding constant nor the number of sites of substance P receptors in corneal epithelial cells, suggesting that the synergistic effect was not regulated at the receptor level. Various extracellular signals activate the intracellular signal transduction system, thus amplifying specific biological functions. We found that the addition of inhibitors of protein kinase C or tyrosine kinase clearly inhibited the synergistic effect of substance P and IGF-1 on corneal epithelial migration, demonstrating that protein kinase C and tyrosine kinase are involved in the synergistic effect. During corneal epithelial wound healing, epithelial cells must attach to a provisional, extracellular fibronectin matrix. We previously reported that interleukin 6 and epidermal growth factor (EGF) facilitate corneal epithelial wound healing by activating the expression of fibronectin receptor (integrin). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that substance P and IGF-1 increased expression of mRNA for integrins alpha 5 and beta 1 in cultured corneal epithelial cells and also increased the number of cells that attached to a fibronectin matrix. These findings strongly suggest that substance P and IGF-1 synergistically increase corneal epithelial migration by activating the expression of integrin. Tachykinins share a five amino acid sequence, phenylalanine-free amino acid-glycine-leucine-methionine amide (FXGLM), at the C-terminus. Studying substance P, we found that a four amino acid sequence at the C-terminus, FGLM, was the minimum amino acid sequence for the synergistic effect on corneal epithelial migration. Structurally similar tetrapeptides mimicking other members of the tachykinin family isoleucine-glycine-leucine-methionine amide (IGLM), valine-glycine-leucine-methionine amide (VGLM), tyrosine-glycine-leucine-methionine amide (YGLM), and the tripeptide glycine-leucine-methionine amide (GLM) did not have any synergistic effect with IGF-1. Based on these findings in vitro, we investigated the effect of eye drops containing substance P plus IGF-1 or FGLM plus IGF-1 on the epithelial wound closure of rabbit corneas in vivo. Both combinations significantly facilitated corneal epithelial wound closure. In a clinical setting, the administration of substance P plus IGF-1 effectively treated corneal epithelial defects in a patient with Riley-Day syndrome, a disease in which corneal epithelial defects persist because of loss of corneal sensation and hypolacrimation. In a patient with neurotrophic keratopathy due to trigeminal nerve paralysis following surgery, eye drops containing FGLM plus IGF-1 eliminated superficial punctate staining. (ABSTRACT TRUNCATED)

Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea.

We find that substance P (SP) and insulin-like growth factor-1 (IGF-1) demonstrate a synergistic effect on the stimulation of rabbit corneal epithelial migration in an organ culture. The addition of either SP or IGF-1 alone did not affect epithelial migration, while the combination of SP and IGF-1 stimulated epithelial migration in a dose-dependent fashion. The synergistic effects of SP and IGF-1 on corneal epithelial migration were nulled by the addition of a SP antagonist or enkephalinase. Among neurotransmitters (vasoactive intestinal peptide, calcitonin gene-related peptide, acethylcholine chloride, norepinephrine, serotonin) or tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin), only SP demonstrated a synergistic effect with IGF-1 on cellular migration. In contrast, the combination of SP and IGF-1 did not affect the incorporation of 3H-thymidine into corneal epithelial cells. The attachment of the corneal epithelial cells to fibronectin, collagen type IV, and laminin matrices increased after treatment of the cells with SP and IGF-1, but SP or IGF-1 by themselves did not affect the attachment of the cells to these extracellular matrix proteins. An identical synergistic effect on corneal epithelial migration was observed when an NK-1 receptor agonist was used in place of SP, suggesting the synergistic effect of SP and IGF-1 might be mediated through the NK-1 receptor system. These results suggest that the maintenance of the normal integrity of the corneal epithelium might be regulated by both humoral and neural factors.

Idiopathic retroperitoneal fibrosis.

We treated a patient with idiopathic retroperitoneal fibrosis accompanied by right ureteral constriction. Pyelography, ureterography, and abdominal CT scan were pertinent diagnostics. Close collaboration between the surgeon and the urologist is required when attempting to treat such patients.