New isolation methods and polymerase chain reaction strain discrimination techniques for natural products screening programs

1995 ◽  
Vol 73 (S1) ◽  
pp. 946-954 ◽  
Author(s):  
Toru Okuda ◽  
Mieko Yanagisawa ◽  
Fumihiro Fujimori ◽  
Yuri Nishizuka ◽  
Yuki Takehana ◽  
...  
Keyword(s):  
Density Gradient ◽  
Random Amplified Polymorphic Dna ◽  
Gradient Centrifugation ◽  
Fungal Spores ◽  
Dilution Method ◽  
Isolation Method ◽  
Screening Programs ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
The Difference

A new isolation method of fungi was established by using the Percoll density gradient centrifugation. We found that the difference in the specific gravity allowed us to separate mixtures of fungal spores of Aspergillus, Fusarium, Listeromyces, Penicillium, and Trichoderma. When this technique was applied to soil samples, a wider variety of fungi were isolated compared with those obtained from an ordinary serial dilution method. For screening purposes, it is necessary to eliminate redundant strains from the strains initially isolated. For the efficient discrimination of strains, the random amplified polymorphic DNA (RAPD) method was applied. The establishment of a standard method for RAPD enabled us to minimize the deviation of Rf values between gels to less than 0.03. Conversion of the Rf values to binary matrices provided a matrix data that could be analyzed statistically. Based on the RAPD, it was possible to select genetically distinct fungal strains within a species. Key words: fingerprinting, isolation method, microbial screening, PCR, Percoll density gradient, RAPD.

Purification of dispersed rat adrenal zona glomerulosa cells by Percoll density gradient centrifugation and the isolation of a population of cells highly responsive to adrenocorticotrophin

1986 ◽  
Vol 109 (3) ◽  
pp. 351-NP ◽  
Author(s):  
F. W. Chu ◽  
P. J. Hyatt
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Sephadex Column ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Glomerulosa Cells ◽  
Unit Gravity Sedimentation

ABSTRACT Percoll density gradient centrifugation is a simple, inexpensive and convenient method to eliminate contaminating zona fasciculata (ZF) cells from unpurified rat adrenal capsular glomerulosa (ZG) cell preparations (with less than 0·1% ZF cells in the final cell preparation). Basal steroid (aldosterone and corticosterone) output by the purified (PG) cells was unchanged. These purified cells, although free from ZF contamination, were more highly responsive than expected to ACTH (3 nmol/l). When PG cells were further separated by Sephadex column filtration, the filtered PG cells exhibited the steroidogenic response of ZG cells purified by unit gravity sedimentation and Sephadex column filtration, i.e. reduced basal steroid output and an ACTH response reduced to that stimulated by K+ (8·4 mmol/l). Although the cells retained in the column resembled the filtered PG cells ultrastructurally, they showed unchanged basal steroid output and a high ACTH response with increased latepathway activity (the conversion of corticosterone to aldosterone). By combining Percoll density gradient centrifugation and Sephadex column filtration we have a method for the isolation and study of both the high-and low-response rat ZG cells which are free from ZF contamination. J. Endocr. (1986) 109, 351–358

scholarly journals Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations

1994 ◽  
Vol 304 (2) ◽  
pp. 617-624 ◽  
Author(s):  
J C Osypiw ◽  
R L Allen ◽  
D Billington
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Rat Hepatocytes ◽  
Marker Enzymes ◽  
Cytotoxic Effects ◽  
Percoll Density Gradient Centrifugation ◽  
Cell Layers ◽  
Density Band ◽  
Percoll Density Gradient

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

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