Biomedical subjects
Publications and source records attributed to C W Gehrke.
A major glutamine tRNA from rat liver was purified. Post-labeling techniques showed its nucleotide sequence to be: pG-G-U-U-C-C-A-U-m(1)G-G-U-G-psi-A-A-D-Gm-G-D-D-A-G-C-A-C-U-C-U-G-G-A-Cm-U-C-U-G-A-A-psi-C-C-A-G-C-G-A-U-m(5)C-m(5)C-G-A-G-psi-psi-C-A-m(1)A-A-U-C-U-C-G-G-U-G-G-A-A-C-C-U-C-C-A(OH).Images
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It was known for some time that cancer patients excrete in their urine elevated levels of modified nucleosides. From earlier work, we were able to show that most of these modified nucleosides originate from transfer RNA (tRNA). The modifications are achieved at the macromolecular level by enzymes after primary synthesis. Such modifications are highly specific and, therefore, when the modified nucleosides accumulate from the tRNA breakdown, they cannot be reinserted randomly by the polymerases and must be excreted. We found that the modifying enzymes are aberrantly hyperactive in every malignant tissue. We also found that there is abnormally high turnover of tRNA in malignant tissues, which is probably the source of the elevated levels of excretion products. Since these products originate from a cardinal component of the molecular biology of every cell, the determination of markers in the urine may be a universal indicator of malignancy. We are focusing on the use of these markers in syndromes whose diagnoses are otherwise difficult. Since the marker levels return to normal very soon after chemotherapy, such determinations can be used to monitor the effectiveness of therapy. Therefore, the clinical oncologists can adapt their protocols to the specific need of a patient.
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.
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.
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.
A sensitive, selective, pre-column derivatization method was used with high-performance liquid chromatography to measure norepinephrine, dopamine and serotonin in plasma from normal and malignant hyperthermic (MH) pigs. Samples were carefully collected from control and stressed animals under halothane anesthesia. Using a simple extraction method involving pre-column derivatization with o-phthaladehyde and ethyl acetate partitioning, the samples were chromatographed in less than 50 min. Norepinephrine was found to be elevated in MH pigs as the syndrome progressed, reaching levels eight-fold greater than control pigs under anesthesia. These experiments provide some evidence for our hypothesis that a failure to metabolize excess norepinephrine may be one of the key metabolic defects in causing the pathophysiology of the malignant hyperthermia-stress syndrome. The application of our chromatographic method in animal and human tests may provide a pattern of biogenic amine types and levels that could be diagnostic in identifying susceptible humans and carrier animals.
As a primary feasibility study for the selection of biomarkers for more extended clinical evaluation, 17 potential biomarker candidates were measured in the body fluids of patients with ovarian carcinoma. For comparative purposes, patients were staged and separated into three groups: those considered completely free of disease, those with residual but minimal tumor, and those with advanced disease. Individual markers included plasma carcinoembryonic antigen, serum human chorionic gonadotrophin, urinary beta-aminoisobutyric acid, serum UDP-galactosyltransferase, and urinary hydroxyproline. Structurally related groups of biomarkers included six modified nucleosides and three polyamines analyzed in urine, and three serum protein-bound neutral carbohydrates. The respective chromatographic methods developed for these latter biochemical materials enabled all the individual compounds in each group to be quantitated simultaneously in one analytical run. The general frequency and degree of elevation for the total number of biomarkers was directly proportional to increasing tumor burden with specific exceptions, human chorionic gonadotropin and beta-aminoisobutyric acid. Galactosyltransferase was the most frequently elevated in the limited disease categories. Several of the biomarkers were elevated in the majority of patients with advanced disease and appeared potentially superior to carcinoembryonic antigen or human chorionic gonadotrophin.
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.
Saccharomyces cerevisiae synthesizes one major tryptophan transfer ribonucleic acid (tRNATrp) species (isoacceptor A) carrying characteristic base modifications. Under tryptophan limited growth conditions wild-type strain X2180-1A exhibited a second important tRNATrp species (isoacceptor B). The total amount of tRNATrp, approximately 2.5 pmol (mg dry wt)-1, stayed essentially constant during amino acid shift-down experiments. The amount of isoacceptor B relative to total tRNATrp was 10 to 15% during amino acid sufficient exponential growth conditions, but increased during tryptophan limitation three- to four-fold. Analysis of the base compositions showed that isoacceptor B differed from isoacceptor A in one respect only: 2'-O-methylguanosine, a modified guanosine base occurring at position 17 of the major isoacceptor A tRNATrp, was not detectable in hydrolysates of purified isoacceptor B. The biological significance of isoacceptor B is discussed.
The monitoring utility of serial patterns of a single marker (carcinoembryonic antigen) level and multiple marker (three carbohydrates-fucose, mannose and galactose) levels in patients with small cell carcinoma of the lung was investigated using a quantitative approach. A serial multiple regression (SMR) model was formulated to assess the disease response following the first to the tenth course of therapy and resulted in the multiple correlations ranging from 0.183 (NS)-0.706 (P less than 0.005) for CEA, and 0.502 (P less than 0.250)-0.760 (P less than 0.025) for three carbohydrates, respectively. The appraisal of these markers utilizing the SMR model points out that: (1) the serial levels of CEA greater than 5.0 ng/ml are significantly correlated with the disease course whereas the serial levels less than 5.0 ng/ml reflect the trend of variation in the disease course, but with less accuracy; and (2) the levels of three carbohydrates, any one elevated before and during therapy, are significantly correlated with the disease course.
Ten tumor markers were measured in serum or urine at approximately three month intervals in patients with breast cancer following mastectomy but before development of overt metastatic disease. In 23 patients who later had metastases, only three markers, alkaline phosphatase, carcinoembryonic antigen (CEA), and gamma-glutamyl transpeptidase (gamma-GT) were consistently abnormal prior to the development of detectable metastases in more than one patient. In half the patients, a "lead interval" of three months or more was obtained using these three markers and little advantage was obtained by the addition of any other biochemical marker. The value of these three measurements was then assessed in a larger group of patients and compared with other tests for metastases. Alkaline phosphatase, CEA, gamma-GT, clinical examination, and chest x-ray were the best indices of the metastatic state in breast cancer, being collectively abnormal in 98% of patients at first presentation with metastases. The authors recommend screening patients postoperatively with these five tests for metastases; more detailed tests should only be carried out if results of one or more these are abnormal.
The use of a selected group of biological markers in breast cancer patients who are disease free but at risk of relapse after mastectomy has potential for detecting recurrent tumor before there is clinical evidence. In this preliminary study, multiple materials were serially analyzed in the body fluids of patients without overt tumor but receiving adjuvant chemotherapy because of positive axillary nodes at surgery. The total frequency of elevated levels was determined and compared for those patients who remained disease free, for those who subsequently relapsed, and for a third group of patients with proven metastases. Frequency of elevation was directly proportional to increasing disease. Although differences in the relative frequency of individual materials was observed, the same trends with increasing tumor burden were found. The results suggest that the serial measurement of biological markers has potential for indicating the presence of occult disease. A nucleus of biological markers to be considered should include carcinoembryonic antigen, urinary hydroxyproline/creatinine ratio for bone lesions, and gamma-glutamyltranspeptidase for liver involvement.
A minor modification in the automated analytical system of the official AOAC semiautomated method for determining crude proteins results in an automated method for determining urea and ammoniacal nitrogen in animal feeds and their ingredients. Urease enzyme which has high activity, yields a clear solution in water, has low ammonia impurity, and is inexpensive is used in the automated method. Weights from 1 to 2.5 g feed sample are dissolved in water, and sample solutions are analyzed at the rate of 40 samples/h. Five AAFCO feed check samples were analyzed repeatedly by the automated method, and results were compared with the grand averages from the check sample reports. The official AOAC manual urease method was used by AAFCO participants. Average recovery of urea and ammoniacal nitrogen was 100.6% by the automated method relative to the AAFCO reported averages. The range of recoveries as 98.5-102.7%. The non-protein nitrogen (NPN) concentrations, expressed as protein equivalent, ranged from 3.40 to 63.04% protein on these samples. The average relative standard deviation for the automated analyses was 0.77%, compared with 1.54% for the manual method. This method is an important adjunct to laboratories using or considering use of the semiautomated method for crude protein and needing further information on NPN.
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.
Sodium bisulfite is a mutagen which can specifically deaminate more than 96% of the cytosine residues in single-stranded DNA via formation of a 5,6-dihydrocytosine-6-sulfonate intermediate. Under the same reaction conditions, only 2-3% of the 5-methylcytosine (m5Cyt) residues in single-stranded XP-12 DNA, which has 34 mole% m5Cyt, was converted to thymine (Thy) residues. In contrast, at the deoxynucleoside and free base levels, the same treatment with bisulfite and then alkali converted 51% and > 95%, respectively, of the m5Cyt to the corresponding Thy derivatives. However, the rate of reaction of m5Cyt and its deoxyribonucleoside was much slower than that of the analogous quantitative conversion of cytosine or deoxycytidine to uracil or deoxyuridine, respectively. The much lower reactivity of m5Cyt and its derivatives compared to that of the unmethylated analogs is primarily due to a decrease in the rate of formation of the sulfonate adduct.
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.