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K C Ehrlich

Publications and source records attributed to K C Ehrlich.

51 records · Page 3Linked to original sources
PubMed1987

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes.

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.

Bacteriophages↗
PubMed1987

Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes.

The Fusarium mycotoxin, 4-deoxynivalenol, is an abundant, natural contaminant of corn and wheat. 8-Oxo-12,13-epoxytrichothecenes related to 4-deoxynivalenol were synthesized; they either lacked the 7-hydroxyl but contained a hydroxyl at C-4 (7-deoxynivalenol) or lacked substituents at C-3 and C-7 (3,7-dideoxynivalenol). The ability of these synthetic analogs and their acetylated derivatives to inhibit protein synthesis by cultured mammalian cells was compared to that of 4-deoxynivalenol. Whereas the 50% inhibitory dose (ID50) for murine erythroleukemia cells was about 1 microgram/ml for 4-deoxynivalenol and 3,7-dideoxynivalenol, all of the other analogs were at least 10-fold less potent. When tested at their ID50 dose, all of the 8-oxotrichothecenes, except 4-deoxynivalenol and 3,7-dideoxynivalenol, caused polysome 'run-off', indicating that, at this dose, they are inhibitors of polypeptide chain initiation. With 4-deoxynivalenol and 3,7-dideoxynivalenol, polysomes remained at control levels indicating that these toxins prevent polypeptide chain elongation. From these results and comparisons to previous studies of 8-oxo-12,13-epoxytrichothecenes (trichothecolone, trichothecin, nivalenol and fusarenone X), trichothecenes with substituents at both C-3 and C-4 predominantly inhibit polypeptide chain initiation, whereas those lacking one substituent at either site are inhibitors of chain elongation.

Cells, Cultured↗
PubMed1986

Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.

Base Sequence↗
PubMed1986

Developmental stage specificity and dose response of secalonic acid D-induced cleft palate and the absence of cytotoxicity in developing mouse palate.

Incidence of cleft palate (CP) in full-term mouse fetuses was evaluated following administration of 25 mg/kg of the mycotoxin, secalonic acid D (SAD), to groups of female mice on each of Days 10, 11, 12, 13, 14, or 15 of pregnancy. Although the highest numerical incidence (45.3%) of cleft palate resulted following SAD exposure on Day 12 of pregnancy, and the response tapered off to 16.9% on Day 10 and 0% on Day 15 of pregnancy, similar responses were produced also following exposures on Days 11 (38.4%) and 13 (39.9%) of pregnancy. Maternal exposure to doses of 0, 15, 20, 25, or 30 mg/kg of SAD, given on Day 12 of pregnancy indicated that although fetuses in the 30-mg/kg group had the highest incidence (51.9%) of CP, the effect was associated with increased resorptions and decreased fetal weights. The 25-mg/kg dose was optimally teratogenic (45.3% cleft palate) and maximally tolerable with neither an increase in resorptions nor a decrease in fetal body weights. Cytotoxicity of the optimally teratogenic dose of SAD (25 mg/kg given ip) on Day 12 of pregnancy was evaluated as a possible mechanism of SAD teratogenicity using indices such as mesenchymal cell density, mitotic index, and the uptake of [3H]thymidine in the developing palatal shelves. No evidence of SAD cytotoxicity was obtained in palatal shelves indicating a possible role for nonlethal cellular effects of SAD in the pathogenesis of CP. These studies also suggest the suitability of the maternal 25-mg/kg dose of SAD to study cellular biochemical effects in the developing embryo without the complicating influence of cytotoxic effects.

Animals↗
PubMed1986

Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs.

Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.

Base Sequence↗
PubMed1985

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol.

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.

Animals↗
PubMed1984

Naphtho-gamma-pyrone production by Aspergillus niger isolated from stored cottonseed.

Aspergillus niger was found to be the predominant fungal contaminant of stored cottonseed. Seven strains were isolated and grown on rice. The hexane-insoluble material from methylene chloride extracts of 2-week-old cultures contained components toxic to mice. Based on high-pressure thin-layer and liquid chromatographic analyses, the major components in the mixture were eight different naphtho-gamma-pyrones. Of these, the hydrated dimeric naphthopyrones aurasperones B and C occurred in higher yield than aurasperones A, iso-A, and D and the monomeric naphthopyrones flavasperone and rubrofusarin, all of which were present in the mixture. In addition, fonsecin monomethyl ether was isolated. This metabolite may be a precursor in the biosynthesis of the hydrated aurasperones; it has not been identified previously as a metabolite of A. niger. The relative amounts of the different naphthopyrones were dependent on both the growth substrate and the fungal isolate.

Aspergillus niger↗
PubMed1984

Simple method for isolation of 4-deoxynivalenol from rice inoculated with Fusarium graminearum.

A new method for preparative isolation of 4-deoxynivalenol (DON) is presented. This method avoids the loss of material during purification on silica gel by column chromatography. DON and 3-acetyldeoxynivalenol in crude extracts of rice inoculated with Fusarium graminearum were converted to triacetyldeoxynivalenol; the acetylated product was easier to purify by silica gel chromatography than DON is. After hydrolysis and further purification on a charcoal-alumina column, the 71% pure DON was recovered in yields as high as 450 mg of DON per kg of rice. Subsequent separation on a Sephadex LH20 column yielded DON that was greater than 90% pure.

Chromatography, High Pressure Liquid↗
PubMed1982

Digestion of highly modified bacteriophage DNA by restriction endonucleases.

The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.

Base Sequence↗
PubMed1982

Secalonic acid D: natural contaminant of corn dust.

The mycotoxin secalonic acid D was detected for the first time as a natural contaminant of corn dust obtained from grain storage elevators. Secalonic acid D amounts ranged from 0.3 to 4.5 ppm (0.3 to 4.5 micrograms/g), as determined by reversed-phase high-performance liquid chromatography.

Dust↗
PubMed1981

A novel, highly modified, bacteriophage DNA in which thymine is partly replaced by a phosphoglucuronate moiety covalently bound to 5-(4',5'-dihydroxypentyl)uracil.

Bacteriophage SP-15, which infects Bacillus subtilis, contains a highly modified DNA in which 62% of its thymine residues are replaced by 5-(4',5'-dihydroxypentyl)uracil to which is attached a phosphoglucuronate via a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain. Glucose is also bound to this residue probably by glycosidic linkage to the other hydroxyl group of the pentyl side chain. In 0.3 M KOH at 37 degrees C, glucuronic acid 1-phosphate is slowly released from this DNA. After enzymatic or acid-induced dephosphorylation, this sugar was identified by chromatography in two thin layer chromatography systems, conversion to glucuronolactone under conditions known to lactonize glucuronic acid, and reaction in four colorimetric assays for hexuronic acids. Phage SP-15 DNA is the first DNA found to have a uronic acid moiety or a phosphate which is not part of the phosphodiester backbone. The glucuronic acid phosphate might be derived from uridine pyrophosphoglucuronic acid, whose glucuronic acid moiety is normally destined for synthesis of teichuronic acid in the host cell wall.

Bacillus subtilis↗
PubMed1981

Proteoglycan synthesis by rat lung cells cultured in vitro.

Cells having a fibroblast-like morphology were cultured from explants of adult rat lung tissue. (35S)Sulfate was incorporated into sulfated proteoglycans in the medium at a linear rate for up to 96 h, while the rate of incorporation into the cell layer increased gradually until reaching a plateau at 40 h. The culture medium contained proteoglycans which migrated as a single peak with Kav = 0.10 on Bio-Gel A-15. Their glycosaminoglycan components (Kav = 0.70 on Bio-Gel A-15) contained predominantly chondroitin sulfate (33 to 44% of the total galactosaminoglycans) or dermatan sulfate chains. Based on the results of chondroitinase AC-II and periodate degradation, disaccharide repeating units of the dermatan sulfate were composed of 36% iduronic acid, 50% 2-sulfoiduronate, and 14% glucuronic acid. A similar composition was found for the dermatan sulfate in the cell fraction. Almost one-half of the sulfate label in the cell fraction was in a heparan sulfate proteoglycan which migrated on Bio-Gel A-15 with Kav = 0.30. The heparan sulfate chains (Kav = 0.81 on Bio-Gel A-15) had few, if any, sulfated N-acetylglucosamine residues and did not contain 2-sulfoiduronic acid in neighboring disaccharide repeat sequences. These results indicate that fibroblast-like lung cells synthesize several types of multichain sulfated proteoglycans which have properties in common with those found in lung tissues.

Animals↗
PubMed1978

Artificial capillary perfusion cell culture: metabolic studies.

Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture units containing 80 to 150 fibers reached populations that used 0.073 +/- 0.025 mumol per min glucose and 0.76 +/- 0.26 microliter per min oxygen and excreted 0.078 +/- 0.038 mumol per min lactic acid. From these data it is estimated that these units contain approximately 2 x 10(7) cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not affected significantly by change in flow rate except at perfusion flow rates less than or equal to 0.05 ml per min. The cell population, as measured by metabolic activity, did not increase significantly when the serum content of the medium was less than or equal to 1%. No major differences were found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture, or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of mammalian cells in culture.

Cell Count↗
PubMedDate not supplied

Mycotoxins in grain dust: method for analysis of aflatoxins, ochratoxin A, zearalenone, vomitoxin, and secalonic acid D.

A multimycotoxin method is presented to quantitate aflatoxins, ochratoxin A, zearalenone, secalonic acid D, and vomitoxin in grain dust. Dust spiked with these mycotoxins was extracted sequentially with methylene chloride followed by acetonitrile-water (86 + 14). Vomitoxin was recovered in the latter extract and all other mycotoxins were recovered in the methylene chloride. Aflatoxins and ochratoxin were quantitated by fluorescence measurement on silica thin layer chromatographic plates. The other mycotoxins were quantitated after cleanup by reverse phase liquid chromatography and ultraviolet detection. Recoveries from dust spiked in the parts per billion (ng/g) range were approximately 80% (SD = 15-29%) for all mycotoxins. Minimum detectable amounts ranged from less than 0.5 ng/g for aflatoxins to 20 ng/g for zearalenone.

Aflatoxins↗