Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: probable role of sphingomyelin.

The coelomic fluid (CF) of the earthworm Eisenia foetida exhibits a wide variety of biological activities. We found that the CF was not toxic to 42 species, belonging to seven invertebrate phyla, almost all in aquatic adults and larvae exposed to CF. Eleven teleostean species tested died in 0.2-1% CF mostly between 10 and 120 min and the effects were dose-dependent. Tadpoles of the toad Bufo japonicus formosus died in 0.4-2% CF between 80 and 225 min depending upon size, with larger tadpoles surviving longer. Before dying, all experimental tadpoles developed curled and shrunken tails. The Okinawa tree lizard, soft-shelled turtle, Japanese quail, mouse and rat all died after i.v. injection of CF (above 20 microl/kg). Thus, CF was not toxic to invertebrates, but toxic to vertebrates. After heating, CF lost its toxicity to fish, tadpoles and mice. Both CF and lysenin incubated with sphingomyelin-liposomes (SM-liposomes) were no longer toxic, suggesting the involvement of SM in the toxicity. Lysenin, which is a constituent of CF and known to bind specifically to sphingomyelin, exhibited toxicity similar to that of CF. Thus, lysenin in CF is probably responsible for the toxic effects of CF by binding to SM in vertebrate tissues. The bodies of invertebrates might contain little or no SM, while those of vertebrates do contain SM. The coelomic fluid of the earthworm Pheretima communissima has no toxicity to mouse.

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida.

Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.

Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida.

Lysenin, a novel protein that we isolated from the coelomic fluid of the earthworm Eisenia foetida, binds specifically to sphingomyelin (SM) among various phospholipids found in cell membranes, and causes cytolysis. The plasma membrane of mammalian spermatozoa is known to contain SM at relatively high levels and we therefore examined the effects of lysenin on the spermatozoa of various animals. Lysenin had lethal effects on spermatozoa of 5 of 33 species of invertebrates tested and on spermatozoa of 30 of 39 species of vertebrates. We postulated that plasma membranes of the spermatozoa of most invertebrates might not contain SM whereas those of most vertebrate species might contain SM. These possibilities were supported by our failure to detect SM chemically in the testes of three species of invertebrates, in none of which spermatozoa responded to lysenin. In contrast, we detected SM in the testes of all 25 vertebrate species examined, irrespective of a negative or positive response of spermatozoa to lysenin. None of the six species of Protista examined was affected by lysenin. Our survey suggests that, in general, the spermatozoa of animals can be grouped into two categories, invertebrate and vertebrate, depending on the absence or presence of SM in their plasma membrane. The incorporation of SM into spermatozoa seems first to have occurred in protochordates during the course of evolution. Discussions about the exceptional responses to lysenin observed in the spermatozoa of five species of invertebrates and of nine species of vertebrates are made from phylogenetic and reproductive viewpoints. J. Exp. Zool. 286:538-549, 2000.

Lysenin, a novel sphingomyelin-specific binding protein.

Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcoreTM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (kon = 3.2 x 10(4) M-1 s-1) and dissociated extremely slowly (koff = 1.7 x 10(-4) s-1), giving a low dissociation constant (KD = 5.3 x 10(-9) M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.

Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle.

Lysenin, which causes contraction of rat vascular smooth muscle, is a protein that was isolated from the earthworm Eisenia foetida. A cDNA encoding lysenin was isolated by use of a partial cDNA probe that had been generated by the PCR with a primer designed by reference to an internal peptide sequence of lysenin. This clone had an ORF encoding 297 amino acid residues. The amino acid sequence deduced from the cDNA revealed the absence of any significant homology to those of previously characterized vasoactive substances. The recombinant lysenin was produced in Escherichia coli. This protein and native lysenin isolated from the earthworm had similar contractive activities when tested on rat aorta. Northern blot analysis of the RNA from various tissues of the earthworm indicated that lysenin is produced by the coelomocytes.

Single-dose kinetics of primidone in human subjects: effect of phenytoin on formation and elimination of active metabolites of primidone, phenobarbital and phenylethylmalonamide.

Effect of repetitive administration of phenytoin (PHT) on the single-dose pharmacokinetics of primidone (PRM) was investigated in 3 healthy male subjects. The peak concentration of unchanged PRM was achieved at 12 and 8 h after the administration of PRM in the absence and the presence of PHT, respectively. The elimination half-life of PRM was decreased from 19.4 +/- 2.2 (mean +/- S.E.) to 10.2 +/- 5.1 h (p < 0.05) and the total body clearance was increased from 24.6 +/- 3.1 to 45.1 +/- 5.1 ml/h/kg (p < 0.01) in the presence of PHT. No significant change was observed for the apparent volume of distribution between the two treatments. In the absence of PHT, the measurable amount (> or = 0.1 mumol/l) of phenobarbital (PB) and phenylethylmalonamide (PEMA) did not appear in the serum until 5.3 and 1.3 h after the PRM administration, and the peak concentrations of PB and PEMA were achieved at 52 and 36 h, but the concentrations of both metabolites were very low (PB 1.3 mumol/l; PEMA 1.7 mumol/l). In the presence of PHT, within 0.8 and 0.5 h after the administration of PRM, the derived PB and PEMA appeared in the serum. About a 6-fold increase in the peak concentrations of both the metabolites were observed (PB 8.2 mumol/l; PEMA 11.0 mumol/l). No significant changes were observed for the elimination half-lives of both PB and PEMA in the absence and presence of PHT.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinical or experimental use of an anticancer drug-oil suspension and its characteristics].

The anticancer agent, Nimustine, which is a derivative of Nimustine hydrochloride (Sankyo CC, Ltd), was suspended in an oil, Lipiodol, using an ultrasonic suspender and used in experimental animals and human subjects with malignant tumor. The use of Lipiodol facilitates the fluoroscopic demonstration of the site into which the suspension has been injected. The Nimustine-Lipiodol suspension was almost stable in room air over 7 days and diffusion of suspended Nimustine into saline in vitro was still noted 4 weeks later. Remarkable regression of tumor size was observed when the Nimustine-Lipiodol suspension was locally injected into the lesion of Lewis lung cancer subcutaneously inoculated into mice. Moreover, a marked regression of tumor size and improvement of CEA level in serum were also obtained when arterial injection of the Nimustine-Lipiodol suspension was carried out in patients with metastatic liver cancer. Therefore, local or arterial injection of Nimustine-Lipiodol suspension is considered to be effective as a method of cancer targeting therapy.

Therapeutic efficacy of a new cephamycin, MT-141, in compromised mice.

The antibacterial activity of MT-141 against Escherichia coli and Proteus morganii in compromised mice was investigated and compared with that of latamoxef, cefmetazole and cefoxitin. The bactericidal activity of MT-141 in short-term contact with E. coli and P. morganii was markedly enhanced when combined with mouse serum, and the activity of MT-141 was greater than the activities of the three reference drugs. The antibacterial activities of MT-141 in the liver, spleen and kidney of neutropenic and Sarcoma 180 tumor-bearing mice infected with E. coli and P. morganii were superior to the activities of the reference drugs. MT-141 was more potent than cefmetazole and cefoxitin, and similar to latamoxef in potency against systemic P. morganii infection in Sarcoma 180 tumor-bearing mice.

Synthesis and antihypertensive activity of 5-O-substituted derivatives of 5-hydroxypicolinic acid.

5-O-Substituted derivatives of 5-hydroxypicolinic acids were synthesized from nojirimycin and studied for their antihypertensive activity in unanesthetized spontaneously hypertensive rats (SHRs) restrained in wire mesh cage and acute toxicity in mice. 5-n-Butoxy-picolinic acid (ND-186) was found to have antihypertensive activity comparable to fusaric acid and lesser acute toxicity. Introduction of halogeno group, in particular trifluoromethyl group to the omega-position of 5-n-butoxy group resulted in the enhancement of antihypertensive activity. The acute toxicity was also lowered 3-5 times compared to that of fusaric acid. Replacement of alkyl group with phenyl group resulted in a slight increment of activity. Some of ester derivatives of ND-186 potentiated the antihypertensive activity and reduced the acute toxicity. There was good correlation between partition coefficient (log P) of ester groups and their antihypertensive activities; compound with higher lipophilicity showed higher antihypertensive activity under the condition of oral administration. However, esterification of other compounds such as 5-halogenoalkoxy- and 5-(substituted) phenoxypicolinic acid was not accompanied with activity increment. Considering from the balance of the antihypertensive activity in SHRs and the acute toxicity in mice, 5-(5',5',5'-trifluoropentoxy)-picolinic acid was selected as a candidate for further evaluation.

Analgesic effect of novel organogermanium compound, GE-132.

A novel organogermanium compound, Ge-132, carboxyethylgermanium sesquioxide, showed enhancement of 0.5 mg/kg morphine analgesia in both administration routes of oral administration (p.o.) and intraperitoneal injection (i.p.) in the Tail-Flick test, and the effect was completely abolished by 0.5 mg/kg Naloxone, stereospecific opiate antagonist. Ge-132 alone, 250 mg/kg i.p., did not show any antinociceptive action by assessing the Tail-Flick test and the Hot-Plate test. By the intracerebral injection of Ge-132, 100-1000 micrograms, prolongation of Tail-Flick latency was observed and the action was abolished by 50 micrograms CaCl2 injection. Although bestatin which is reported to enhance the morphine analgesia inhibits enkephalinase and enkephalin aminopeptidase, Ge-132 did not show any inhibition on both enkephalin degrading enzymes. The possibility for the mode of action of Ge-132 was discussed.

Relationship between enhancement of morphine analgesia and inhibition of enkephalinase by 2S, 3R 3-amino-2-hydroxy-4-phenylbutanoic acid derivatives.

The effects of nineteen AHPA* derivatives were examined on morphine analgesia by tail-flick test in rats and on enkephalinase inhibition which was based on the formation of tyrosyl-glycyl-glycine from met-enkephalin. The correlation between the enhancement of morphine analgesia in vivo and enkephalinase inhibition in vitro was analyzed. The different analogs varied considerably in the degree of enhancement of morphine analgesia and inhibition of enkephalinase. A close relationship between enkephalinase inhibition expressed by IC50 in vitro and enhancement of morphine analgesia in vivo was observed in thirteen out of nineteen AHPA derivatives examined. One of other six AHPA derivatives which showed weak effectiveness in potentiating on morphine analgesia but was highly potent as an enkephalinase inhibitor, caused potent analgesic action when it was applied intracisternally indicating poor penetration of the blood brain barrier. The possibility was discussed that some of other compounds excluded from the linear relationship might act on other enkephalin degrading enzymes such as aminopeptidase.

[Physiological effects and antitumor efficiency of ACNU in postoperative patients with stomach cancer--an evaluation of the treatment combined with FT-207 or 5-FU dry syrup].

In 26 patients with cancer of the stomach, bone marrow function, cell mediated immunity and plasma CEA level were examined after administration of ACN-U. ACNU was given intravenously and intermittently with 5-FU given orally daily. Results 1) Delayed myelosuppression was observed in every cases and reached to the nadir in 2-7 weeks after ACNU administration. 2) Bone marrow suppression and recovery therefrom were observed earlier in platelet counts than in RBC and WBC counts. 3) Suppression of cell mediated immunity was not observed later than 4 weeks after ACNU administration. 4) Plasma CEA level decreased or stopped to increase after ACNU administration in 5 out of 8 patients having a high CEA level before treatment. 5) Regression of tumor size was observed in 4 out of 5 patients in whom a tumor was palpable. Conclusion ACNU has a strong antitumor activity and may be more effective if combined with antimetabolite agent, such as 5FU. Total dosage of ACNU given safely in one series was considered to be 150-200mg. Next series of ACNU administration should be started after restoration of platelet counts more than 100,000/mm3 was obtained.

In vitro and in vivo antibacterial activity of 9,3"-Di-o-acetyl midecamycin (Mom), a new macrolide antibiotic.

9,3"-Di-O-acetyl midecamycin (Mom) showed in vitro antibacterial activity against clinical isolates of Staphylococcus aureus and was effective against some of the strans resistant to erythromycin (Em) and josamycin (Jm). The distribution pattern of the level of drug-sensitivity of the isolates to Mom was similar to that of Jm but different from that of Em. Mom- or Jm-resistant strains gradually or rapidly developed among S. aureus strains, that were sensitive or resistant to other macrolide antibiotics. There was no significant difference between Mom and Jm as regards the rate of development of resistant strains. These mutants were always resistant to both Mom and Jm. Mom, like Jm, was effective against Em-inducible strains of S. aureus. The in vivo study demonstrated that Mom was more potent than Jm, similar in its potency to Em against systemic staphylococcal infections, and that it was effective against the infection due to an Em-resistant clinical isolate of S. aureus. Mom was more effective than either Jm or Em against a staphylococcal kidney infection in mice.

Pharmacological analysis of the mechanism of action of phenthiazamine, a piscine anesthetic.

Phenthiazamine was developed by Sekizawa et al. as a centrally acting anesthetic for fish. Frog sympathetic ganglion was used as a model to elucidate the mechanism of its anesthetic action. The positive ganglionic potential was enhanced immediately after application; then, the potential and the late negative ganglionic potential were markedly reduced by an anesthetic concentration of this compound. The hyperpolarization caused by 1 mM dopamine was enhanced by phenthiazamine. This enhancement of dopamine hyperpolarization and of the positive ganglionic potential may coincide with the fact that cyclic adenosine monophosphoric acid (cAMP) phosphodiesterase was inhibited by the compound as shown in our previous paper. Guinea-pig ileum contraction elicited by electrical field stimulation and by dimethyl-phenyl-piperadinium (DMPP) was reduced by a similar concentration of this compound, while the contraction elicited by acetylcholine and methacholine was not inhibited. The inhibition of contraction elicited by electrical field stimulation and DMPP may thus due to inhibition of acetylcholine release by this compound. The inhibition of ileum contraction by this compound was reversed by higher doses of Ca2+ (5.5 mM). The time required to reduce the positive ganglionic potential in the sympathetic ganglion by phenthiazamine was prolonged in the presence of higher concentrations of Ca2+. The Ca2+-dependent action potential of guinea-pig ureter was reduced by this compound, whereas it did not affect the Na+-dependent action potential.