Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2003-10-01
2004-11-16
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S253500, C540S472000
Reexamination Certificate
active
06818422
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel K97-0239 substance useful for prevention and treatment of arteriosclerosis and diseases caused therewith by specifically inhibiting generation of the foamy macrophage and production thereof.
2. Description of Related Arts
It is recently known that the life-style related diseases such as hyperlipidemia and obesity of adults are progressed to pathological conditions such as atherosclerosis, myocardial infarction, cerebral hemorrhage and cerebral apoplexy connecting directly with death. At present, statin series drugs such as pravastatin, fluvastatin, cerivastatin and atorvastatin are used as drugs for prevention and therapy of arteriosclerosis. These drugs have an effect for reducing cholesterol levels in blood by inhibiting HMG-CoA reductase, one of rate-limiting enzyme in cholesterol biosynthesis in vivo. However, arteriosclerosis is caused by complicated and complex etiology and drugs having different mechanism of action are strongly required.
In early lesion of atherosclerosis, machrophages infiltrating into the arterial endothelium recognize the denatured low density lipoprotein (hereinafter sometimes designates as LDL), which is generated by some denaturation such as oxidation and glucosylation of LDL, in the blood stream, incorporated endlessly and hydrolyze to generate free cholesterol and fatty acid, which are converted into cholesterol ester and triacylglycerol and accumulated in cytoplasm as lipid droplets. Then the macrophages are converted to foam cells to develop arteriosclerosis. Consequently, substances, which inhibits a process for forming the foamed macrophage, is expected to directly suppress development of arteriosclerotic lesion, but drugs, including statin series drugs, having such effect have not been known.
In such the actual condition, to provide substance, which inhibits the process for forming the foamed macrophage, is expected to contribute to the human health as a novel drug for directly acting arteriosclerotic lesion and suppressing the progress of disease.
An object of the present invention is to provide the novel K97-0239 substance satisfying such the expectation and the process for production thereof.
An another object of the present invention is to provide the drug for prevention and treatment of arteriosclerosis comprising K97-0239 substance as an active ingredient and the microorganism for production of said substance.
SUMMARY OF THE INVENTION
We have screened substance having inhibitory action for forming the foamy macrophage on metabolites produced by microorganisms, and found that a strain
Streptomyces
K97-0239, which was newly isolated from soil, produced, in the culture liquid, the substance having activity for inhibiting formation of the foamy macrophage. Then the substance having activity for inhibiting formation of the foamy macrophage was isolated and purified from said cultured mass, and we have found that the substance having such chemical structure has never known previously and designated the substance as K97-0239 substance.
The present invention has completed based on such knowledge, and relates to K97-0239 substance comprising K97-0239A substance represented by the following formula [I],
and K97-0239B substance represented by the following formula [II],
The present invention further relates to a process for production of novel K97-0239 substance comprising culturing a microorganism belonging to genus
Streptomyces
and having ability to produce K97-0239 substance, accumulating K97-0239 substance in a cultured mass and isolating K97-0239 substance from said cultured mass.
The present invention further relates to
Streptomyces
sp. K97-0239 which is the microorganism belonging to genus
Streptomyces
and having ability to produce K97-0239 substance.
The present invention still further relates to a microorganism which is
Streptomyces
sp. K97-0239 FERM BP-7514.
The microorganism having ability to produce K97-0239 substance represented by the above formula [I] and [II] (hereinafter designates as “K97-0239 substance producing microorganism”) belongs to genus
Streptomyces
, and, for example, a strain
Streptomyces
sp. K97-0239, which was newly isolated from a soil sample collected in the Setagaya-ku, Tokyo by us, is an example of the strain used most effectively in the present invention.
Taxonomical properties of the strain K97-0239 are as follows.
(I) Morphological Properties
Vegetative mycelia grow well on various agar media and no fragmentation is observed. Aerial mycelia are abundantly grown on glycerol asparagine agar medium and inorganic salts-starch agar medium and show grayish color. On microscopic observation, chains of more than 20 spores were observed on the aerial mycelia, and the morphological form straight chain and size of spore is 0.7-0.9×1.0-1.3 &mgr;m with cylindrical form. Surface of the spore is smooth. No sclerotia, sporangia and zoospore are observed.
(II) Properties on Various Media
Culture properties of the producing strain of the present invention determined by the method of E. B. Shirling and D. Gottlieb (International Journal of Systematic Bacteriology, 16: 313, 1966) are shown in the following. Color tone was determined referring to Color Harmony Manual, 4th Ed. (Container Corporation of America Chicago, 1958) as a standard color, and color name as well as attached code number in the parenthesis. Unless otherwise noted, results are observation of cultures at 27° C. for 2 weeks on various media.
Culturing properties
Sucrose-nitrate agar medium
Growth
good growth, pearl (3ba)
Reverse side
pearl (3ba)
Aerial mycelium
abundant, beige gray (3ih)
Soluble pigment
none
Glucose-asparagine agar medium
Growth
moderate growth, fresh pink (3ca)
Reverse side
fresh pink (3ca)
Aerial mycelium
none
Soluble pigment
none
Glycerol-asparagine agar medium (ISP)
Growth
good growth, pastel orange (4ic)
Reverse side
pastel orange (4ic)
Aerial mycelium
abundant growth, ashes (5fe)
Soluble pigment
none
Starch-inorganic salt agar medium (ISP)
Growth
good growth, cream (2ca)
Reverse side
ivory (2db)
Aerial mycelium
abundant growth, lead gray (5ih)
Soluble pigment
none
Tyrosine agar medium (ISP)
Growth
moderate growth, fresh pink (3ca)
Reverse side
fresh pink (3ca)
Aerial mycelium
moderate growth, orchid tint (10ba)
Soluble pigment
none
Oatmeal agar medium (ISP)
Growth
good growth, light melon yellow (3ea)
Reverse side
light tan (3gc)
Aerial mycelium
abundant growth, ashes (5fe)
Soluble pigment
none
Yeast-malt extract agar medium (ISP)
Growth
good growth, light amber (3ic)
Reverse side
light amber (3ic)
Aerial mycelium
abundant growth, lead gray (5ih)
Soluble pigment
none
Nutrient agar medium
Growth
good growth, ivory (2db)
Reverse side
ivory (2db)
Aerial mycelium
none
Soluble pigment
none
Peptone-yeast-iron agar medium (ISP)
Growth
good growth, dusty peach (3ec)
Reverse side
dusty peach (3ec)
Aerial mycelium
none
Soluble pigment
none
Glucose-nitrate agar medium
Growth
poor growth, pearl (3ba)
Reverse side
pearl (3ba)
Aerial mycelium
none
Soluble pigment
none
Grycerol-calcium malate agar medium
Growth
good growth, dusty peach (3ec)
Reverse side
dusty peach (3ec)
Aerial mycelium
moderate growth, orchid tint (l0ba)
Soluble pigment
none
Glucose-peptone agar medium
Growth
moderate growth, light wheat (2ea)
Reverse side
light wheat (2ea)
Aerial mycelium
none
Soluble pigment
none
Physiological Properties
(1) Formation of melanin pigment
(a) Tyrosine agar medium
negative
(b) Peptpone-yeast-iron agar medium
negative
(c) Tryptone-yeast liquid
negative
(d) Simple gelatin medium (21-23° C.)
positive
(2) Nitrate reduction
positive
(3) Liquefaction of gelatin (21-23° C.)
negative
(simple gelatin medium)
(4) Starch hydrolysis
positive
(5) Coagulation of defatted milk (37° C.)
positive
(6) Peptonization of defatted milk (37° C.)
positive
(7) Growth temperature
10-38° C.
(8) Utilization of carbon sources (Pridham-Gottlieb agar
medium)
Utilize
glucose, arabinose
Slightly utilize
fructose, sucrose
Not utili
Omura Satoshi
Tomoda Hiroshi
Gakkou Houjin Kitasato Gakuen
Lilling Herbert J.
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