Biomedical subjects

C W Gehrke

Publications and source records attributed to C W Gehrke.

At least 37 records · Page 2Linked to original sources

PubMed1987

High-performance liquid chromatographic-radioimmunoassay method for the measurement of angiotensin II peptides in human plasma.

A highly selective high-performance liquid chromatographic-radioimmunoassay method for the measurement of individual endogenous angiotensin peptides in human plasma is described. This method allows the complete resolution of the immunoreactive angiotensin II peptides. We have also measured the angiotensin peptide levels and compared them in both pooled and individual human plasma. The effects of inhibition of angiotensin-converting enzyme on the angiotensin peptide levels have also been observed in a patient with renovascular hypertension with the plasma angiotensin II level being reduced greater than seven-fold. This new methodology was validated by recovery experiments in plasma over a range of physiological levels using two methods of detection, radioimmunoassay and liquid scintillation counting. Consistent recoveries near 80% have been achieved for each peptide in plasma at concentrations over a physiological range. The described method enables the direct measurement of the circulating angiotensin peptides and the elucidation of their specific roles in physiological and disease states.

Angiotensin II↗

PubMed1987

In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome.

Functional 30S ribosomes were reconstructed from total Escherichia coli 30S ribosomal proteins and 16S ribosomal RNA synthesized in vitro by T7 RNA polymerase. Up to 700 mol of RNA/mol of template could be obtained. The transcript lacked all ten normally modified bases and had three additional 5' G residues, an A----G change at position 2, and, in 22% of the molecules, one or two extra 3' residues. The synthetic 16S RNA could be assembled into a particle that cosedimented with authentic 30S and was indistinguishable from 30S by electron microscopy. When supplemented with the 50S subunit, the particles bound tRNA to the 70S P site in a codon- and Mg2+-dependent manner. The specific binding activity was 94% that of particles reconstituted with natural rRNA and 52% that of native 30S. Cross-linking to P site bound tRNA was also preserved. Changing C-1400, the residue known to be close to the anticodon of P site bound tRNA, to A had little effect on reconstitution, but the C----G substitution caused a marked inhibition of assembly. tRNA could bind to both reconstituted mutants, but cross-linking was greatly reduced. These results show that none of the modified bases of 16S RNA are essential for P site binding and that position 1400 may be more important for ribosome assembly than for tRNA binding. Base-specific in vitro mutagenesis can now be used to explore in detail the functional properties of individual residues in ribosomal RNA.

Base Sequence↗

PubMed1987

N4-methylcytosine as a minor base in bacterial DNA.

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.

Bacteria↗

PubMed1986

Quantitative measurement of mRNA cap 0 and cap 1 structures by high-performance liquid chromatography.

Viral and eukaryotic mRNA molecules have a unique 5'-end. The 5'-terminus consists of m7G(5')ppp(5')N'(m)pN''(m), which is termed a "cap" structure. The study of these cap structures has led to the development of many methods of identification and analysis. Many of the methods have been time-consuming or have not been able to distinguish between the different caps, and they are quantifiable only by employing radiolabels. This paper presents the use of reversed-phase high-performance liquid chromatography as a rapid and efficient tool for the separation, identification and quantitation of caps. An ion-exchange enrichment procedure was also developed for the isolation of cap 0 and cap 1 structures from unfractionated RNAs. The recoveries of different caps ranged from 83 to 99%, with a relative standard deviation range of 1.3-4.4%. In this method, caps were released from commercially obtained rabbit globin mRNA by nuclease P1 digestion. The products of digestion were treated with alkaline phosphatase and separated on an octadecylsilyl column using stepwise or gradient elution. Cap structures and any internal modified nucleosides were identified by their retention times and UV spectra relative to reference compounds. The amount of each cap 0 or cap 1 structure was determined by its UV absorbance relative to a known quantity of reference compound. This method allows the quantitation of 0.2 nmol or more of cap 0 and cap 1 structures. Total UV spectra can be obtained for 0.5 nmol or more of cap. This methodology permits investigations on viral and eukaryotic mRNA cap biosynthesis and turnover during viral transformation, differentiation, cap synthesis in the cell cycle, etc.

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↗

PubMed1986

DNA cytosine methylation and heat-induced deamination.

The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37 degrees C and pH 7.4 were approximately 9.5 X 10(-10) and 2.1 X 10(-10) sec-1, respectively. N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.

5-Methylcytosine↗

PubMed1985

Plasma levels of norepinephrine and epinephrine during malignant hyperthermia in susceptible pigs.

Malignant hyperthermia (MH) is a genetic disease of man, swine, dogs, cats, and horses. The syndrome is normally triggered by inhalational anesthetics or the administration of depolarizing muscle relaxants such as succinylcholine or various environmental stress factors. We have used the MH-susceptible pig as an animal model to study the hormonal changes developing during this highly lethal syndrome. High-performance liquid chromatography with electrochemical detection was used for the quantitation of the plasma levels of norepinephrine and epinephrine during MH. This research presents evidence that the rapid release of massive quantities of norepinephrine (up to 108 ng/ml) into the blood stream occurs simultaneously with the initiation of tachycardia which is the herald signal of the onset of MH. Norepinephrine levels exceed epinephrine by a 4:1 ratio early in the syndrome. Even pigs with MH which do not develop the muscle rigor phase have high levels of circulating norepinephrine. Tachycardia, pulmonary hypertension, increased venous oxygen desaturation, and increasing core temperature develop as the syndrome progresses.

Animals↗

PubMed1985

Pattern of undermethylation of the major satellite DNA of mouse sperm.

Enzymatic hydrolysis and base analysis by high performance liquid chromatography showed that mouse satellite DNA had 30-50% less 5-methylcytosine in sperm than in somatic tissue (1.59 mols % vs 2.40-3.11 mols %). Maxam-Gilbert sequencing and analysis of the intensity of the cytosine bands indicated that the level of methylation of the eight CpGs of the consensus sequence in sperm satellite DNA ranged from 0 to about 50%, considerably lower than the levels reported in somatic tissues. The Mn1I site containing one of these CpGs was cut much more extensively in satellite DNA from sperm than from liver, confirming the undermethylation of this site in sperm DNA.

5-Methylcytosine↗

PubMed1985

DNA methylation is not increased in mouse-human somatic cell hybrids.

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.

Animals↗

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

Mouse mammary tumor virus DNA methylation: tissue-specific variation.

Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpaII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpaII site in Mtv-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.

5-Methylcytosine↗

PubMed1984

Effect of temperature stress on circulating biogenic amines in bovine.

A sensitive, simple and selective chromatographic method using high performance liquid chromatography was developed to measure circulating levels of histamine (HI), norepinephrine (NE), dopamine (DA) and serotonin (5-HT) as indicators of response to thermal stress in two breeds of cattle. Duplicate exposures for 8 hr to 13 degrees C resulted in significantly elevated plasma NE and DA in longhorns but not in Herefords and suggest a relatively greater sensitivity to the cold (13 degrees C) in the longhorn. Environmental temperatures of 32 and 42 degrees C significantly increased rectal temperatures of both breeds with much higher rectal temperatures in Herefords. The 32 and 42 degrees C effects on circulating NE and DA of Herefords were highly significant but not in the longhorn. However, heat stress significantly elevated HI and 5-HT in the longhorn which may account for their relatively greater heat tolerance based on their lower rectal temperatures.

Animals↗

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↗