Biomedical subjects
Publications and source records attributed to E Schauenstein.
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The SH content of the soluble proteins (nanomol./mg protein) from five transplantable rat hepatomas and from the DENA-hepatoma were determined with dithionitrobenzoate (Ellman reagent). Both the total number of thiols as well as the number of SH groups that can be blocked by hydroxypentenal (HPE) increase with increasing growth rate of the tumors. In comparison with the protein thiol content of the slowest growing DENA-hepatoma (doubling time 100 days), the total protein thiols of the fastest growing Yoshida hepatoma (doubling time 2,5 days) increase by 100% and the HPE-sensitive protein thiols by 350%. The total protein thiols are significantly correlated with the growth rate (probability of error 5%), the HPE-sensitive thiols are correlated with high significance (probability of error less than 1%). These results are in accordance with the "Molecular Correlation Concept" of G. Weber and might possibly be understood as a consequence of reprogramming of gene expressions.
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The IgG fraction was isolated from freshly taken blood serum of health persons (male and female) by column chromatography on QAE-Sephadex. After 30 min incubation with DTNB (5,5'-dithio-(2,2'-dinitro)-benzoate) the average photometrically determined quantity of thio-anions was 0.24 +/- 0.02 SH/mole IgG. Since this result remained unchanged even after 24 h incubation with DTNB, interaction with masked thiol groups cannot be assumed. If, however, the serum was incubated with DTNB for 24 h and the IgG fraction then isolated and treated with thioglycolate, an average of 1.51 +/- 0.39 moles of thio-anions were liberated per mole of IgG. This indicates that on 24 h interaction with IgG, DTNB not only reacts with free SH groups, but also opens S-S bonds by a disulfide exchange reaction. The amount of thio-anions resulting from such opened disulfide bonds was calculated as the difference between 1.51 0.24, i.e., 0.64 S-S/mole IgG. This would be accounted for if approximately 54% of the IgG fraction is composed of a subfraction containing 1 labile disulfide bone per mole. The average of 1.51 thio-anions per mole IgG resulted from 130 single values with an essentially normal statistical distribution and standard deviation well within the usual biological range.
The usefulness of protein films denaturated and fixed with ethanol/ether as cytochemical model systems was investigated. Kinetics of staining with amido black 10B were analyzed as well as the spectroscopic properties of the protein-dye complex. The absorption coefficient of the complex was estimated. Data obtained so far are compared with both stained cells and the protein-dye complex in solution. Discussion focuses on the influence of fixation and the possibilities to use the films as systems for calibrating cytochemical stainings.
The continuous administration of physiological doses of the branched-chain amino acids leucine, isoleucine, and valine (Leu-Ile-Val) to Yoshida sarcoma-bearing rats caused a significant increase in the survival time by 32% and a significant reduction of tumor size after 3 weeks of growth by 33%. The shift of the nitrogen balance to negative values during the cachectic stage was delayed but not prevented. On the average, less nitrogen (-47 mg/day) were lost by Leu-Ile-Val treated rats compared with untreated tumor-bearing animals (-91 mg N/day). It appeared that Leu-Ile-Val increased the synthesis of carcass proteins, while it left the proteolysis rate unchanged, since the excretion of urea and creatinine was unaffected by these amino acids. The daily excretion of alpha-ketoglutarate, which is correlated with tumor size during the early stage of growth, was decreased during the first 2 weeks by Leu-Ile-Val, but remained for a longer period on a high level than in untreated tumor bearers. The results point to an improvement of the metabolic resistance against carcass protein depletion of the tumor-bearing host by the administration of branched-chain amino acids.
A 4-year survey is presented on the alpha-ketoglutarate (KG) values in whole blood of 200 patients with malignant neoplasms mainly of the gastrointestinal tract and the female breast. A group of patients with benign surgical diseases served as control. KG showed an association with the extent of the primary tumor classified according to the TNM-scheme. The percentages of single values above the 2s-range of the control were as follows: T2: 18%,T3: 41%, and T4: 64%. The mean value of KG had highly significantly increased already in stage T2 as compared to the control group. Rats bearing the Yoshida sarcoma showed a significant correlation between the tumor size and the daily excretion of KG into urine during the early stage of tumor growth. The results suggested that KG cannot be regarded as an early tumor marker in humans, but may be of some value as an aid for the differential diagnosis in advanced tumor stages.
To elucidate the origin of increased concentrations of alpha-ketoglutarate (KG) in tumor bearers the tissue distribution of KG together with the related metabolites citrate, succinate, malate, and glutamate was determined in tumor, liver, gastrocnemius muscle, and blood of rats bearing the solid Yoshida sarcoma and of tumor-free rats. The sum of these metabolites was significantly increased in host liver and blood, respectively, compared with the corresponding tissues of normal rats. Among single metabolites glutamate and malate were significantly increased in host liver. The absolute concentrations were highest in host liver with the exception of KG, which was highest in the tumor. This was taken as indicative for the tumor as the prime source of increased KG in blood of tumor-bearers. No significant metabolic deviations were found in gastrocnemius muscle. The concentration of KG in this muscle of both normal and host animals correlated significantly with that of glutamate. In the tumor the concentration of KG correlated significantly with that of citrate plus succinate plus malate. This type of correlation was absent in liver and muscle of both normal and host animals. Moreover, no correlation existed between KG and glutamate either in liver or in tumor. It was suggested that the metabolic flux through the citric cycle determines the concentration of KG in the tumor.
Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.
In aqueous solution Amido Black B (ASB) forms stable and well-defined complexes with bovine serum albumin (RSA) at pH 5.5. The complexes can be separated by column chromatography. The formation of the complexes consist in a fast reaction during which, after 3 to 5 h approximately, 3 molecules of ASB have been bound per molecule RSA, and of a much slower reaction which, even after a laps of 24 h, is still far from approaching its final stage. With solid films of RSA, after denaturation with ethanol, fast reaction is found to approach its final stage after 10 min reaction time. With these model protein preparations, the molar extinction coefficient of the ASB-protein complexes can be determined: the soluble ASB-RSA complexes can be brought to complete dissociation at pH 12.3. After the additivity of the specific absorptions of both RSA and ASB had been proven, it was possible to determine the content of the solution of ASB and RSA, and therefrom the molar extinction coefficient of the ASB-RSA-complex at Ph 5.5: epsilon 620 = 110,000. ASB-stained ethanol-fixed RSA films show an epsilon 620 of approximately 96,000, if their thickness and specific weight are known. After incubation in watery or ethanolic/TCA solutions of ASB, also animal cells fixed with ether-ethanol show the ASB absorption band to be in the region of 600 nm after removal of the surplus of ASB by thorough washings. As already observed with the RSA films, the kinetics of the staining of the cells show the fast reaction reaching its final stage already after 15 to 20 min. When alcoholic solution of ASB is used, the extinctions are found to be twice or three times higher than those achieved by an aqueous one. After standardization of the staining procedures with both solvents the total extinctions of EATZ, YATZ, rat hepatocytes, chicken thymus and bursa cells were measured and plotted against the macroscopically determined protein content of the respective cells. Highly significant positive linear correlations resulted with staining both in watery and alcoholic solutions, respectively. From the slope of the straight lines, specific extinction coefficient of ASB stained cellular proteins could be calculated up to epsilon' 620 = 1.76 with watery ASB solution and epsilon' 620 = 3.83 with the alcoholic solvent. The soluble ASB-RSA complexes have an epsilon' 620 = 1.67 the ASB stained ethanol denaturated films of RSA an epsilon' 620 of within a range of 1.21 to 1.80.
The increase of plasma cortisol in patients with tumors of five different sites compared with a control group of patients with benign surgical diseases amounted to: +39% (breast), +34% (stomach), +86% (intestine), +60% (skin) and +194% (gall bladder). The first detectable increase of cortisol occurred in patients with tumors classified T 2 according to the TNM scheme (+27% above the control). Highly significant increases were observed for T 3 (+82%) and T 4 (+77%) patients. Patients with palpable lymph nodes showed a most significantly increased cortisol mean value compared with patients without palpable lymph nodes. Similarly, the cortisol mean value of patients with distant metastases was significantly higher than the corresponding value of tumor patients without distant metastases. The question remains open, whether the primary site, the extent of the tumor or the occurrence of metastases is the main determinant for the cortisol increase.
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4-Hydroxyalkenals react easily with SH groups in enzymes and structural proteins to give stable adducts in which the aldehyde is bound through a thioether linkage to the protein. This reaction represents the mechanism by which these compounds block certain metabolic processes (DNA, RNA and protein synthesis, respiration, glycolysis) and exert their antitumour activity. A highly significant inverse correlation exists between the number of hydroxypentenal-sensitive SH groups in the soluble proteins and the doubling time of 12 transplantable animal tumours. Hydroxyalkenals applied peritumourally completely prevent or significantly delay growth of the solid forms of four animal tumours. The almost non-toxic adducts of 4-hydroxypentenal-cysteine 1:1 and crotonal-cysteine 1:2 applied intraperitoneally prevent or inhibit growth of Ehrlich ascites tumour in mice. The possible formation of unsaturated aldehydes in vivo from lipids, carbohydrates and diamines is discussed.
Smears taken from eight probands with carcinoma in situ or invasive carcinoma of the cervix uteri have been stained with DDD and Fast Blue B. The extinctions microspectrometrically measured at 560 nm are directly proportional to the quantity of protein-SH-groups. The extinctions of the total cell (Eges) and of the cell nucleus (EK) are measured in 67 basal cells (BAS), 78 dysplatic cells (DYS), 122 undifferentiated cancer cells (UNIF) and 89 differentiated cancer cells (POLY). From BAS through DYS and UNIF to POLY EK increases by a total of 176%. In all four cell types investigated, linear correlations between EK and Eges have been found to occur with a probability of over 99%. The straight lines ascertained represent a relation between EK and Eges which is obviously very characteristic for each cell type, and it becomes apparent that the measuring points corresponding to each single cell are in each instance so close to the straight line that in most cases a differentiation of the three pathological cell types is possible even without a morphological criterion. The straight lines corresponding to BAS, DYS and UNIF start from a common origin, whereas the straight line corresponding to POLY branches off from the UNIF line only. This is in accordance with the formal genesis of pathological variants observed in the cervical squamous epithelium or in differentiated carcinomas of the squamous epithelium respectively.
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Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.
Evidence is presented that beer contains the following isomerric trihydroxy acids: 9,12,13-Trihydroxy-10 trans-octadecenoic acid (G1), 9,10,13-Trihydroxyl-11 trans-octadecenoic acid (G2), and 9,10, 11-Trihydroxy-12 trans-octadecenoic acid (G3). The compounds were isolated from a CHCl3: MeOH extract of beer by column chromatography and their structures determined by comparison of their mass spectra, Rf-values (TC) and retention times (GC) with the data obtained with the reference compounds G1, G2, and G3 prepared by enzymic oxidation of linoleic acid with barley flour. For the quantitative determination a CHCl3: MeOH extract of 100 ml acidified beer was methylated, silylated and separated by GC. Depending on the specific beer source, the beer contained 4.7--9.0 mg/1 G1, 1--2.4 mg/1 G2 and 0.4--0.7 mg/1 G3. The mechanism of the formation of these compounds from linoleic acid is discussed as well as their possible contribution to the stale flavor of beer which develops upon prolonged storage.
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