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Biomedical subjects

K C Kuo

Publications and source records attributed to K C Kuo.

At least 37 records · Page 2Linked to original sources
PubMed1985

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.

5-Methylcytosine↗
PubMed1984

Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA.

Improved, highly accurate high-performance liquid chromatographic methods for the measurement of the major and modified nucleosides in enzymatic digests of DNA using a single column are described. Four high resolution separation protocols (isocratic, binary, ternary and high speed) with specifically improved selectivity for 5-methyldeoxycytidine (m5dCyd) from Ade, dIno and Guo are presented. From a detailed study of the various factors contributing to the precision and accuracy of the measurement, optimized conditions and quantitative protocols were established. The ternary buffer allows for the first time the determination of N6-methyldeoxyadenosine (m6dAdo) in the same chromatographic analysis with the other deoxyribonucleosides. The binary system allows quantitation of the absolute amounts of each ribo- and deoxyribonucleoside as well as the mole % of each as the second buffer elutes 5'dA and the internal standard 8-bromoguanosine. The isocratic system allows precise quantitation of the mole % of each ribo- and deoxyribonucleoside while eliminating the need for buffer change valves, buffer cycling and column re-equilibration. Also, a high-speed isocratic system is described which permits separation of the deoxyribonucleosides in 6 min. The quantitative, enzymatic hydrolysis of DNA was evaluated by comparing a 40-h, three-enzyme system with a 4-h, two-enzyme procedure. The latter protocol proved to be an excellent hydrolysis method. These high resolution liquid chromatography techniques provide the most precise, sensitive and accurate measurement of m5dCyd available, in a straightforward method using as little as 1 microgram of DNA, and have allowed us to demonstrate: the existence of tissue-specific differences in levels of m5dCyd in DNA of humans, monkeys, rats and mice; that m5dCyd levels in DNA change during fetal development; that genomic undermethylation of DNA is correlated with cancer and the presence of m6dAdo in DNA of thermophilic organisms.

Animals↗
PubMed1984

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs.

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.

Anticodon↗
PubMed1983

Urinary polyamines for evaluating the course of disease for patients with small cell carcinoma of the lung.

Clinical correlates with urinary excretion of polyamines were evaluated for 29 newly diagnosed and 35 previously treated patients with small cell carcinoma of the lung (SCC). The frequencies of pretreatment abnormalities were 12 (41%) for putrescine, 18 (62%) for spermidine, and 20 (69%) for spermine. In assessing disease parameters, the combined use of the abnormalities of spermidine and spermine as a discriminant was more effective than that of all three polyamines; it correlated significantly with extent of limited and extensive disease (P less than 0.001), and also resulted in significant separation of survival curves, the median survival of 11 months for both elevated compared to 19 months for neither or only one elevated (P = 0.062). No significant difference was seen in the abnormalities between no metastasis and one metastasis, whereas the frequencies of the abnormalities was highly increased in two or more metastases. The distribution of polyamines determined at regular treatment intervals showed distinctively more elevated patterns in progressive disease than in stable disease or partial and complete responses (P less than 0.01). In order to evaluate therapeutic effects on the relationship between polyamine excretion and tumor regression, correlations between urinary putrescine and spermidine were determined. The values of the ratio of spermidine to putrescine were significantly smaller in responders than in nonresponders (P less than 0.01); and these may be related to smaller tumor mass and higher tumor proliferative activity in responders, and larger tumor mass and lower tumor proliferative activity in nonresponders.

Carcinoma, Small Cell↗
PubMed1983

The 5-methylcytosine content of DNA from human tumors.

The over-all 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the m5C contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.

5-Methylcytosine↗
PubMed1983

Serum protein-bound carbohydrates and small cell carcinoma of the lung. Correlations with extent of disease, tumor burden, survival, and clinical response categories.

The levels for serum protein bound neutral carbohydrates (fucose, mannose, and galactose) were determined at specific intervals for 40 patients with small cell carcinoma of the lung and compared to the corresponding carcinoembryonic antigen (CEA) levels. In pretreatment samples, the frequency of elevation was 92.5% for fucose and 77.5% each for mannose and for galactose. CEA determined in these same samples was elevated (greater than 5 ng/ml) in 45.0%. One or more of the three carbohydrate levels were elevated in pretreatment serum of 95.0% of the patients. The individual frequency of elevation for each carbohydrate was significantly related to initial stage of disease (P less than 0.01). Median survival was significantly longer for patients based on a discriminant of less than 3 carbohydrates elevated in pretreatment samples (25 months) to all 3 elevated (11 months) with P = 0.0302. A single value, termed the biomarker index, was calculated to represent the summation of the individual carbohydrate levels per individual serum sample. The biomarker index was found to be directly correlated with extent of primary disease, number of metastic sites, tumor burden, and clinical response categories assessed at serial time points. For patients with both low Biomarker Index values and normal CEA levels in pretreatment samples, an initial rise in both determinations occurred frequently corresponding to partial or complete tumor response. The occurrence of such discordant results must be considered as a likely possibility for those patients with low or normal pretreatment biological marker levels and subsequent response to primary chemotherapy.

Blood Proteins↗
PubMed1983

Tissue-specific differences in DNA methylation in various mammals.

The only naturally occurring modified base in vertebrate DNA is 5-methylcytosine. Using a precise high-performance liquid chromatographic analysis of DNA enzymatically digested to deoxynucleosides, we have shown that rats, mice and four types of monkey display tissue-specific as well as species-specific differences in the extent of methylation of their cytosine residues. Several similarities in the patterns of tissue-specific DNA methylation in these mammals and in the previously studied human samples were observed. Compared to most other types of DNA examined, brain and thymus DNAs were hypermethylated which suggests that this hypermethylation is a determinant or a necessary byproduct of mammalian differentiation. In all of the studied rodents and primates, the highly repeated DNA sequence fraction was more methylated than the moderately repetitive or single copy fractions. The tissue-specific differences in overall DNA methylation showed no correlation with what is known about average cell turnover rates nor with the percentage of the genome that is transcribed. Liver regeneration in the rat following partial hepatectomy did not detectably alter 5-methylcytosine levels in liver DNA. A considerable increase in the extent of methylation of total liver DNA was observed during normal development of the rat. The latter phenomenon may be due to a major change in the cellular composition of the liver.

5-Methylcytosine↗
PubMed1983

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.

5-Methylcytosine↗
PubMed1982

Biological markers and small cell carcinoma of the lung: a clinical evaluation of urinary ribonucleosides.

Five minor base ribonucleosides, primarily degradation products of transfer ribonucleic acid (tRNA), were evaluated as potential biological markers for patients with small cell carcinoma of the lung. The urinary concentration for pseudouridine, 1-methyladenosine, 1-methylinosine, N2-methylguanosine, and N2,N2-dimethylguanosine was determined by means of reversed-phase high performance liquid chromatography and quantitatively expressed as a function of creatinine excretion. Comparisons were made with carcinoembryonic antigen (CEA) plasma levels. The total frequency of elevated values for the five nucleosides in pretreatment urine samples was directly related to stage of disease with 24/60 (40%) determinations increased in 12 patients with limited disease and 69/85 (81%) in 17 patients with extensive disease. For these same patients, CEA levels were elevated respectively in 2/11 (18%) of the former and 9/17 (53%) of the latter group. The frequency and degree of elevation of the nucleoside/creatinine ratios in pretreatment samples from patients with extensive disease was correlated directly with increasing number of metastatic sites. Of the five nucleosides, the mean number elevated was two for limited disease, 3-4 for extensive disease with one metastatic site, 4 for two or three, and 5 for four or more sites of metastases. Based on a summation of pretreatment nucleoside/creatinine ratios, a discriminant for survival was derived giving curves separating patients (P = 0.086) similar to the discriminant based on stage of disease. Although discordant results were noted, an overall correlation of 75% agreement with clinical assessment was estimated in response categories when monitoring changes associated with therapy.

Antineoplastic Agents↗
PubMed1982

Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides.

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme--substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis--HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

Chromatography, High Pressure Liquid↗
PubMed1982

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA.

5-methylcytosine residues in DNA underwent deamination at high temperatures. Furthermore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [14C]-5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [14C] 5-methylcytosine residues. Upon incubation at 95 degrees C in a physiological buffer or at 60 degrees C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those on cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.

5-Methylcytosine↗
PubMed1982

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.

5-Methylcytosine↗
PubMed1982

Modified ribonucleosides as biological markers for patients with small cell carcinoma of the lung.

A variety of individual modified ribonucleosides may be elevated in the urine of cancer patients. They can be readily measured quantitatively in a single reversed-phase high-performance liquid chromatographic run. A total of 41 patients with small cell carcinoma of the lung were studied. For 5-ribonucleosides determined in the pretreatment urine of 28 patients, the respective frequency of elevation was directly related to stage of disease. One or more nucleosides were evaluated in the pretreatment urine of 27 out of 28 patients (96%). Included were 11 patients with limited disease and 10 (91%) had 2 or less than 2 nucleosides elevated, whereas 16 out of 17 (94%) with extensive disease had 3 or more elevated. Based on this same discriminant, median survival was significantly extended for patients with 2 or less nucleosides elevated (24 months) in contrast to 3 or more (10 months). Using a single number to represent the summation of equally weighted individual nucleoside values as a composite score, a direct relationship was found between increasing extent of disease or tumor burden. This was in contrast to more variable results for carcinoembryonic antigen analyzed in plasma samples obtained at the same time. When determined serially the composite score paralleled in general the clinical response categories for individual patients.

Carcinoembryonic Antigen↗
PubMed1980

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.

We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1, Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 microgram of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.

Alkaline Phosphatase↗
PubMed1980

Biologic markers in breast carcinoma: clinical correlations with urinary polyamines.

Urinary polyamine levels were evaluated in patients with breast carcinoma. The individual levels of putrescine, spermidine, spermine, and cadaverine, and the product/precursor levels of putrescine, spermidine, and spermine were analyzed. Elevations of one or more individual polyamines or of the ratios were found in 50% of patients with metastatic disease, 38.5% of preoperative patients, and 35.7% of 5--24 week postoperative N + patients. Sequential sampling of patients with metastatic disease suggested that changes in elevated polyamine levels tend to reflect the clinical course of the disease, especially for the association of treatment failure with rising elevated values. The presence of one or more elevated parameters prior to treatment of metastatic disease tended to be associated with a higher response rate (85.7 vs. 68.4%) than all normal levels. Five of nine patients who recurred postoperatively had preceding postoperative polyamine elevations. In addition, there was a trend for a shorter disease-free time among patients with one of more elevated polyamine parameters between 5--24 weeks postoperatively than among patients with normal parameters. These data suggest that measurement of urinary polyamine levels, including calculation of the product/precursor levels, may be a useful clinical adjunct in the management of patients with breast carcinoma.

Breast Neoplasms↗