Increased melanogenesis in cultured epidermal melanocytes from patients with neurofibromatosis 1 (NF 1).

Melanocyte cultures from the normally pigmented skin of patients with neurofibromatosis 1 (NF 1) have a higher melanin content than those from the skin of healthy donors. An additional increase in the amount of melanin per cell was found in 5 out of 6 lines of melanocytes derived from café au lait macules of NF 1 patients. Omission of the tumor promoter phorbol-12-myristate-13-acetate from the culture medium brings about a comparable increase in the melanin content in all three kinds of melanocyte cultures. Cultures of NF 1 melanocytes show a higher tyrosine hydroxylase activity than those of control melanocytes, and incorporate larger amounts of dihydroxyphenylalanine than the latter. We conclude that melanogenesis in epidermis melanocytes is affected by defective alleles of the NF 1 gene. Our findings do not contradict the hypothesis that the defect underlying NF 1 impairs the inhibition of a wild-type RAS oncogene by interfering with the GTPase-activating function of the NF 1 gene product.

Preparative free solution isoelectric focusing of human erythrocyte uroporphyrinogen I synthase in an ampholyte pH gradient.

A free solution electrofocusing method for uroporphyrinogen I synthase (EC 4.3.1.8) in an Ampholine pH gradient on a preparative scale is described. Partial purification of the enzyme was achieved in a 4-h focusing run. Enzyme activity was found in the pH range of pH 5.1 to pH 7.0. Complete separation of the most basic and most acidic isozyme from the control and the acute intermittent porphyria (AIP) patient was obtained in this single-step procedure. The level of enzyme activity has been shown to be reduced to about half the normal value in erythrocytes of two patients from a family with AIP. A shift of maximal activity toward the acidic side of the pH gradient was observed with the abnormal enzyme. In contrast to the normal isozyme set with seven isozyme bands, the fluorescence of the three basic bands and the second acidic band was greatly reduced, whereas the intermediate forms showed increased fluorescence intensity.

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.

Isolation of the low-molecular-mass form of catechol O-methyltransferase from rat liver and immunocytochemical localization of the enzyme in the glycogen compartment.

The low-molecular-mass form of two distinct catechol O-methyltransferase activities (S-adenosyl-L-methionine: catechol O-methyltransferase, COMT, EC 2.1.1.6) has been purified to homogeneity from rat liver using 40-70% ammonium sulfate precipitation, gel filtration on Sephadex G-100, adsorption on hydroxyapatite C and ion-exchange chromatography on DEAE-Sepharose CL-6B. The relative molecular mass Mr, determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis is 22 400 +/- 500. Irradiation of the enzyme in the presence of 8-azido-[methyl-3H]AdoMet results in the specific labeling of the catalytic site of the enzyme. Photolabeling was successful with crude COMT preparations and with the isolated enzyme. Immunocytochemical studies present new information about the localization of the low-molecular-mass form in the liver parenchyma. Subcellularly COMT immunoreactivity could be attributed exclusively to the compartment with glycogen granules. Nucleus, mitochondria and endoplasmic reticulum showed no immunostaining.

Immunocytochemical demonstration of catechol methyltransferase in Candida tropicalis.

Catechol methyltransferase (S-adenosyl-L-methionine : catechol O-methyltransferase; EC 2.1.1.6) was localized immunocytochemically in the yeast Candida tropicalis by the unlabelled antibody enzyme method, involving soluble peroxidase-antiperoxidase complex. Immunoreactivity was detected by light and electron microscopy in the outer layer of the cell wall and at the plasma membrane. The possible function of the methyltransferase in C. tropicalis id discussed.

Immunological studies of catechol methyltransferase from the yeast Candida tropicalis.

Immunization of rabbits with purified catechol methyltransferase from Candida tropicalis yielded a potent antiserum. Ouchterlony double diffusion analysis showed a single precipitin line. There was no cross reactivity with the catechol methyltransferase from rat and bovine liver. Specific antigen-antibody interaction was demonstrated by a potent inhibitory effect of the antibody on the yeast enzyme. Immunological titration and quantitative precipitin reaction of the enzyme showed that the maximum amount of precipitable complex occurred at the equivalence point where enzyme activity was completely inhibited.

The influence of estradiol and adriamycin on the RNA biosynthesis in breast cancer.

The extent of the relationship between hormone dependence and cytostatica sensitivity of the tumors was examined for primary breast cancers under standardized in vitro conditions. The effects of estradiol and adriamycin on the RNA biosynthesis in the tumor cells were compared on the basis of the 3H-uridine incorporation in a short-term test system. Estradiol particularly influenced the RNA metabolism of higly differentiated carcinomas, whereas adriamycin exhibited the greatest inhibitory effect on the RNA synthesis in the tumor cells of undifferentiated carcinomas. An exact, unequivocal separation of the breast cancers investigated into hormone-sensitive, cytostatica-resistant and hormone-independent, cytostatica-sensitive tumors, however, was not possible under the in vitro conditions chosen.

[Cytostatic effects on nucleic acid synthesis in primary tumors and lymph node metastases of human breast cancer (author's transl)]. / Nukleinsäuresynthese und deren Beeinflussung durch Cytostatica bei Primärtumor und Lymphknotenmetastase von Mammacarcinomen.

Nucleic acid synthesis and influence of cytostatic agents (adriamycin, cyclophosphamide) were investigated in 73 cases of human breast cancer and in 19 cases of correlating lymph node metastases. An interrelationship between nucleic acid synthesis, proliferation and tumor stage could not be proven at the time of the operation. The labeling index, expressing the proliferation of tumor cells, and the incorporation of radiolabeled nucleic acid precursors (3H-thymidine, 3H-uridine) as an indicator of nucleic acid synthesis were higher in the investigated lymph node metastases than in the primary tumor. Nucleic acid synthesis was suppressed in primary and metastatic lesions in the same way by cytostatic agents.

Purification and properties of a catechol methyltransferase of the yeast Candida tropicalis.

In an effort to investigate catechol methyltransferase activity in sources other than mammalian tissues and cells, a high level of enzyme activity was found in the yeast fungus Candida tropicalis CBS 94. Partial purification of the enzyme (approx. 550 fold with a recovery of 7%) could be achieved by using ion-exchange and gel filtration techniques. The molecular weight was estimated at 32,000 +/- 2,000 by gel filtration on Sephadex G-100. In isoelectric focusing experiments on Sephadex G-75 the enzyme exhibited a pI-value of 5.0 +/- 0.1. In contrast to catechol methyltransferase from various mammalian tissues the enzyme activity was prepared from the pH 5-sediment. The substrate specifity is comparable to other catechol methyltransferases.

Incorporation studies of nucleic acid precursors in gastric cancer: an attempt in individualized chemotherapy.

Measurements of the rate of incorporation of radioactively labeled nucleic acid precursors into the DNA and RNA of gastric carcinoma cell suspensions indicated variable rates of proliferation for the tumors. The rate of incorporation generally correlates to the cytological level of differentiation of the carcinoma. Reduced differentiation of the tumors showed a corresponding increase in the rate of proliferation. Knowing the proliferation-dependent effect of most cytostatica, this results in a resistance to cytostatica of highly differentiated gastric cancers. The nucleic acid synthesis of proliferatively active tumors could only be partially inhibited by the cytostatica tested (5-fluorouracil, adriamycin). Carcinomas with metabolic possibility for compensation of the active mechanism of the cytostatica were biochemically resistant. Due to the resulting methodical problems and unaccountable patient-dependent causes of resistance, a conclusive statement about cytostatica-sensitive tumors is difficult to make in incorporation studies.

[Effect of estradiol on RNA biosynthesis of human breast carcinoma cells in vitro]. / Einfluss von Ostradiol auf die RNS-Biosynthese von menschlichen Mammacarcinomzellen in vitro.

The possibility of differentiating estrogen-sensitive human breast cancer using incorporation studies with labeled uridine as a precursor of RNA metabolism is described. The purpose of this study was to explore inadequate function of the estrogen receptor as an alternative or supplementary aid in selecting patients for hormonal manipulation. The disadvantage of the test is that only hormone dependence of a proliferating tumor cell population can be evaluated. Highly differentiated breast cancer cells exhibit the greatest estrogen sensitivity. The hormone-dependent tumors of premenopausal women show an increase in RNA synthesis, whereas uridine incorporation appeared to be inhibited in postmenopausal women.

Pretherapeutic cytostatic agent sensitivity testing of breast cancer. / Prätherapeutische Cytostatica Sensibilität-Testung des Mammacarcinoms.

With and without addition of cytostatic agents, incorporation of radiolabeled precursors into the nucleic acid of breast cancer cells was determined in short-term culture. The labeling index, a measure of tumor proliferation, depended largely on cytologic differentiation. Incorporation of radiolabeled precursors in moderately and low differentiated breast cancers was significantly high, whereas only in a part of these tumors was incorporation inhibited by adriamycin and cyclophosphamide. Therefore a group of drug sensitive and metabolic drug resistant tumors could be defined in vitro. Primary resistance against the tested cytostatic agents has been proved in low proliferating and high differentiated breast cancer cells.

[A sensitive fluorometric determination of catechol methyltransferase activity (author's transl)]. / Eine empfindliche fluorometrische Bestimmungsmethode für die Catechol-Methyltransferase

A method for the determination of catechol-methyltransferase activity is described, based on the measurement of fluorometric intensity of 7-hydroxy-6-methoxycoumarin (scopoletin), enzymatically produced by dihydroxycoumarin in the presence of the methyl donor S-adenosylmethionine.