Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1999-09-10
2001-01-09
McKane, Joseph K. (Department: 1626)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S107000, C562S443000, C548S532000
Reexamination Certificate
active
06171829
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel physiologically active substance PF1191 showing an inhibitory activity to kainic acid toxicity and thus being useful as a remedy and a preventive for diseases caused by nervous disturbances or its salt, and a process for producing the same.
BACKGROUND ART
Excitatory amino acids (glutamic acid, aspartic acid, etc.) play important roles as neurotransmitters in mammals. On the other hand, there have been reported findings one after another that abnormal excitation of excitatory amino acid receptors is one of the causative factors of nerve cell death occurring in brain ischemia, head injury, Alzheimer's disease, Parkinson's disease, Huntington's chorea, etc. It is also suggested that abnormalities in these excitatory amino acid receptors participate in the onset of schizophrenia. Under these circumstances, studies have been vigorously made on excitatory amino acid receptor antagonists and agonists.
Regarding substances with antagonism to excitatory amino acid receptors with microbial origin, it is reported that a substance ES-242 is produced by Verticillium sp. ES-242 (J. Antibiotics, vol. 45, 88-93 (1992)).
To develop an antagonist to excitatory amino acid receptors, the present inventors have studied and searched to find a substance originating in a microbial product which is capable of inhibiting the toxicity of kainic acid on brain nerve cells.
It is also reported that kainic acid is toxic to primarily cultured chick telencephalon nerve cells and this toxicity by the excitatory amino acid is expressed via the cystine transporter system and non-N-methyl-D-aspartic acid (NMDA) receptor (Neuroscience Letters, vol. 139, 205-208 (1992)). This toxicity is inhibited by non-NMDA receptor antagonists including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX).
Accordingly, the present invention aims at providing a substance which is capable of inhibiting the toxicity of kainic acid on brain nerve cells and therefore useful as a remedy and a preventive for diseases caused by nervous disturbances.
DISCLOSURE OF THE INVENTION
By using primarily cultured chick telencephalon nerve cells, the present inventors searched microbial products to find out a substance capable of inhibiting the toxicity of kainic acid on brain nerve cells. As a result, they found that a strain belonging to the genus Eupenicillium accumulated a product having an inhibitory activity to kainic acid toxicity in its culture medium. Next, the above product (i.e., the substance PF1191) was isolated and purified from the culture and identified as a novel substance based on its physicochemical properties. As the results of the subsequent studies, it was confirmed that this substance PF1191 has a stronger inhibitory activity to kainic acid toxicity than that of DNQX. The present invention has been completed based on these findings.
Accordingly, the first gist of the present invention resides in the provision of a novel substance PF1191, which is represented by the following formula (I) and involves all stereoisomers thereof, or its salt:
The second gist of the present invention resides in the provision of a process for producing the physiologically active substance PF1191 or its salt which comprises incubating a fungus belonging to the genus Eupenicillium and being capable of producing the substance PF1191, and collecting the thus produced substance PF1191 from the culture.
MODE FOR CARRYING OUT THE INVENTION
The microorganism to be used in the production of the substance PF1191 in the present invention may be an arbitrary one so long as it belongs to the genus Eupenicillium and is capable of producing the substance PF1191. As an example of the microorganism usable in the present invention, a strain
Eupenicillium shearii
PF1191 (hereinafter referred to as the “PF1191 strain”) having been newly isolated from the soil may be cited.
1. Mycological Properties of PF1191 Strain
(1) Characteristics in Incubation
Colonies grow well on Czapek-yeast extract agar and attain 35 to 40 mm in diameter after incubating at 25° C. for 7 days. They are a white to grayish brown, velvety, radial wrinkled and consisting of a thick mycelial layer. A number of ascomata are formed in submerged hyphae. It has a back face in a pale olive color.
Colonies grow well on malt extract agar and attain 18 to 20 mm in diameter after incubating at 25° C. for 7 days. They are pale brown, velvety, flat and consisting of thin mycelial layer. A number of ascomata are formed in submerged hyphae. It has a back face in a pale orange color.
Colonies grow well on oatmeal agar and attains 18 to 20 mm in diameter after incubating at 25° C. for 7 days. They are grayish brown, velvety, flat and consisting of thin mycelial layer. A number of ascomata are formed on the colony surface to give a granular appearance. It has a back face in a pale yellowish brown color.
At an incubation temperature of 37° C., the growth of the strain is inferior in each of the above-described media to the growth at 25° C.
(2) Morphological Characteristics
Cleistochecia are globose to ellipsoidal, yellowish brown and 150 to 350 &mgr;m in diameter. Each peridium is composed of sclerenchyma cells and turns from pseudoparenchyma into sclerotium. They take 3 to 4 weeks for maturation. Asci are globose to ellipsoidal in shape, 6 to 8×5 to 6.5 &mgr;m in size, 8-spored and evanescent at maturity. Ascospores are lens-shaped and 3.0 to 3.5×2.0 to 3.0 &mgr;m in size, each having two equatorial crests and fine projections on the convex surface. Conidiophores are 200 to 600×2 to 2.5 &mgr;m in size and smooth-walled. Penicilli are biverticillate, metulae are 12 to 20×2.5 to 3 &mgr;m in size and 2 to 4 per stipe, and phialides are ampulliform, 7 to 12×2.5 to 3 &mgr;m in size and 5 to 7 per metula. Conidia are ellipsoidal in shape and 2.5 to 3×2 to 2.5 &mgr;m in size and smooth-wallod.
Based on these mycological characteristics, this strain has been identified as
Eupenicillium shearii
belonging to Plectomycetes and named
Eupenicillium shearii
PF1191 strain. In the identification, use was made of “The ascomycete genus Eupenicillium and related Penicillium anamorphs (Studies in Mycology, No. 23)”, Amelia C. Stolk and Robert A. Samson, Centraal-bureau voor Schimmelcultures, 1983.
This PF1191 strain has been deposited as follows.
{circle around (1)} Depository name: International Depository, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology
Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi, 305-8566 JAPAN
{circle around (2)} Deposition Date: Original deposition date: Dec. 2, 1996 Request for transfer: Feb. 20, 1998 (transferred from FERM P-15973 deposited on Dec. 2, 1996)
{circle around (3)} Deposition No. FERM BP-6263.
Similar to other funguses, the PF1191 strain is liable to undergo changes in its properties. Therefore, use can be made in the present invention of, for example, mutants (either spontaneous ones or mutagenized ones) originating in the PF1191 strain, conjugants or gene recombinants, so long as they are capable of producing the substance PF1191.
2. Method for Incubating Substance PF1191-producing Strain
The microorganism capable of producing the substance PF1191 may be incubated in a medium containing nutrients commonly usable by microorganisms. As the nutrients, use can be made of those conventionally employed in the incubation of funguses. Examples of carbon sources usable herein include glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal oils and vegetable oils. Examples of the nitrogen sources include organic matters such as soybean meal, wheat germ, corn steep liquor, cotton seed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate and urea. Furthermore, it is possible to add to the medium inorganic salts capable of providing ions of sodium, potassium, calcium, magnesium, cobalt, chlorine, ph
Sasaki Toru
Seto Haruo
Shin-ya Kazuo
Yaguchi Takashi
McKane Joseph K.
Meiji Seika Kaisha Ltd.
Oswecki Jane C.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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