DNA-binding proteins in the sera of patients with malignant melanoma.

Sera of patients with various malignancies are known to contain DNA-binding proteins (DBP) which are not present in sera of normal individuals. In this paper sera of patients with malignant melanoma (MM) were examined as to whether characteristic DBP are present, too. DBP are isolated by DNA-affinity chromatography and represent 0.5-0.9% of all serum proteins. After separation of the DBP by SDS slab gel electrophoresis no typical DBP is detectable in sera of MM-patients. However, quantitative differences are found in sera of patients in the clinical stages I-III and/or tumor level 3-5: 1. All 9 sera of patients who had clinical signs of MM contain more DBP with molecular weight (mw) of 20,000-24,000 dalton than control sera. However, these DBP are only increased in 30% of the 22 sera from MM-patients who had clinical signs for 13-73 months after tumor excision. 2. All sera of the 10 MM-patients of whom sera were drawn twice after tumor excision at an interval of 7-46 months without clinical signs, showed a reduction of DBP with mw 30,000, 68,000, and 165,000.

Comparison of DNase, DNA-polymerase and RNA-polymerase activities present in the DNA-binding proteins of normal human dermis, epidermis, horny layer and psoriatic scales.

DNA-binding proteins (DBP) of normal human dermis, epidermis, horny layer and psoriatic scales represent a tissue-specific group of mostly nuclear nonhistone proteins. To analyse their function, the different DBP fractions were examined concerning the presence of DNase, DNA-polymerase and RNA-polymerase activities. DBP of normal epidermis and horny layer contain four different DNases. One DNase of both DBP fractions is active only at pH 5.0. Three DNases of epidermal DBP are active at a pH-range from 5.0--8.5, while the corresponding DNases of horny layer-DBP are most active at pH 7.4. Probably these DNases have changed their pH-optimum during keratinisation. DBP of psoriatic scales include no activity of these three DNases and the pH 5.0-DNases seem to have reduced DNA-affinity. Human dermis DBP contain quite another set of four DNases which hardly can be correlated to the DNases of epidermal DBP. DNA-polymerase activities are present in each fraction and derive from different DNA-polymerases. Two DNA-polymerases with pI-values of 4.5 and 9.3 may correspond to beta- and alpha-DNA-polymerase of eukaryotes, respectively. Further activity of proteins which are focussed at pH 6.5--7.2 and 8.2 could be detected. The proteins represent either tissue-specific DNA-polymerases or further thymidine monophosphate incorporating enzymes. Contrary, RNA-polymerase activity could not be enriched from correlating extracts by DNA-cellulose chromatography.

DNA-binding proteins of psoriatic scales III. Biochemical characterisation as non-histone-proteins.

Histone and non-histone-proteins (NHP) are proteins with a specific affinity to DNA; each group is involved in the regulation of gene expression in its own way. To investigate molecular biologic processes at psoriasis, DNA-binding proteins (DBP) were isolated from psoriatic scales. During this work the proportions of histone and NHP of the DBP fractions were examined. Using two different methods (Bio Rex 70-chromatography and isoelectrofocussing), it was found that the isolated DBP contain only acidic proteins. Likewise, the composition of amino acids is comparable with those of acidic, nuclear proteins of other tissues. Thus the isolated DBP represent to its greatest extent acidic, chromosomal NHP, which obviously are derived from the preserved nuclei of the parakeratotic scale layer.

Long-term tissue culture of epithelial-like cells from human skin (NCTC strain 2544). II. Viscosity changes after enzyme treatment.

Human skin epithelial-like cells (NCTC strain 2544) were grown in NCTC 135 medium. Neuraminidase and hyaluronidase were added to the growth medium. Cells were incubated 96 h at 36 degrees C. Growth rate and viscosity of cell suspensions were measured after forming single cells mechanically (mopping). With addition of neuraminidase and hyaluronidase, respectively, the growth rate remains unchanged. With neuraminidase a distinct raise in viscosity was achieved, whereas with hyaluronidase only a small effect was seen. The characteristic structure viscosity is maintained in all forms of the viscosity curves at different shear-rates.

Disc-electrophoretic separation of water soluble proteins from scales of different forms of ichthyosis.

In extracts of scales of different forms of ichthyosis, disc-electrophoretic separation of water soluble proteins was performed. Number and position of the protein bands correspond with number and position of the bands of extracts of normal keratin and psoriatic scales. However, in comparison to normal keratin, all examined forms of ichthyosis showed in zone II the enriched bands Nos. 6 and 7. This permits a distinct differentiation from psoriasis in which in zone II the bands Nos. 9 and 10 are enriched. In congenital ichthyosiform erythroderma and in ichthyosis combined with atopic dermatitis, in zone III the bands, containing gamma globulins, are enriched as an expression of the concomittant exudative process. The protein content of scale extracts of ichthyosis is 2 to 5 times lower than the one of psoriasis.

DNA-binding proteins of psoriatic scales. I. Isolation by affinity chromatography and characterisation by SDS slab gel electrophoresis.

DNA binding proteins (DBP) are shown to be engaged in the regulatory functions of the genome. Since the accelerated epidermopoesis in psoriasis indicates changed gene activities, the examination of DBP possible could help to clarify the pathological processes. Psoriatic scales were used to elaborate the method. DBP was isolated from a protein extract by affinity chromatography with DNA-cellulose according to Alberts et al. About 3-5% of postribosomal crude protein (PCP) extract had an affinity to DNA. The separation of DBP on SDS polyacrylamide slab gels showed a characteristic spectrum of proteins with molecular weights from 12.7 to 150 X 10(3) daltons. The protein band spectrum of the concentrated DBP fraction was different from that of the crude extract. These observations show the isolated DBP to represent an own group of water soluble proteins with DNA binding abilities.

DNA-binding proteins of psoriatic scales. II. DNA-Affinity of the three characteristic proteins increased in the water soluble extract of psoriatic scales.

The disc-electrophoretic pattern of a water soluble protein extract of psoriatic scales contains three characteristic proteins. They appear in higher amounts in psoriatic scales compared to normal human stratum corneum. Assuming that these proteins have any functional implication at DNA, their possible correlation to DNA-binding proteins (DBP) of psoriatic scales was examined. The disc-electrophoretic spectrum of DBP was compared with the protein profile of crude extract. However, no protein of DBP fraction correlated with one of the three characteristic proteins present in a water soluble extract. This signifies these proteins to be more a metabolically enriched product than proteins of chromosomal origin.

[Premature cutaneous porphyria caused by oral contraceptives (author's transl)]. / Porphyria cutanea praematura durch hormonelle Kontrazeptiva

Four women, aged 23 to 26 years, fell ill with cutaneous hepatic porphyria. All of them had taken oestrogen-contaning oral contraceptives three to eight years before the illness. In one instance there was a time relationship with a progestogen preparation. The clinical and biochemical signs were those of porphyria cutanea tarda, but contrary to this disease there was a shift towards heptacarboxyporphyrin in the uroporphyrin/heptacarboxyporphyrin ratio (thin-layer chromatography, classified according to Doss). This difference and the onset at an early age suggest that the described disease may be called a hormone-induced premature cutaneous porphyria.

[Treatment of porphyria cutanea tarda with and without phlebotomy (author's transl)]. / Behandlung der Porphyria cutanea tarda mit und ohne Aderlässe

Extensive investigations form 1962 to 1973 on 23 patients with porphyria cutanea tarda are reported. Two collectives of patients with different treatments are compared. One collective was treated conventionally with "liver therapy, abstinence from alcohol, and rest in bed", the other collective with "phlebotomy, abstinence from alcohol, and rest in bed". Both collectives showed a nearly equal decrease of uroporphyrin excretion. The unfavourable effect of alcohol in this disease was confirmed. In the post-observation period, minimal excretion of uroporphyrin was found to be more frequent and to persist longer in the collective treated by phlebotomy. In this collective, abstinence from alcohol was more consistently adhered to.

Isolation and physicochemical characterisation of the DNA from normal human skin and psoriatic scales.

In psoriatic parakeratosis preserved nuclei are found in the epidermal horny layer. The question arises, whether the components of the nuclei remain unchanged. Thus we isolated and purified the DNA of psoriatic scales and normal skin. Comparing the melting points and circular dichroism of both DNAs no significant difference could be detected. These data indicate the same guanine-cytosine content as well as comparabel helical compositions of the isolated DNAs.

Long-term tissue culture of epithelial-like cells from human skin (NCTC strain 2544). I. Measurement of viscosity.

Viscosities of cell suspensions of human skin epithelium (NCTC strain 2544) were determined by a Wells-Brookfield rotation viscosimeter. A structure viscosity may be postulated by the form of the viscosity curves at different shear-rates. At a cell number of 0.5 X 10(6) cells/ml a viscosity of 1.094 centipoise (25degrees C, shear-rate 230 s-1) could be found. Since a suspension of single cells may be easily formed mechanically (mopping) the NCTC 2544 cells are useful as investigational model regarding the investigation of membrane characteristics.

Separation, isolation, and amino acid composition of the albumin-like fraction from psoriatic scales.

The albumin-like fraction of psoriatic scales was isolated either directly by slab gel electroporesis or after previous treatment with polyethylene glycol6000. After both isolation procedures the amino acid composition was determined. Both proteins were rich of glutamic acid, glycine, and alanine. On the other hand both contained very small amounts of cysteine and tyrosine. Only after the direct isolation procedure the amino-sugar galactosamine could be detected.

Subcellular distribution of phosphatases, proteinases, and ribonucleases in normal human stratum corneum and psoriatic scales.

The subcellular distribution of phosphatases, proteinases, and ribonucleases of normal human stratum corneum and psoriatic scales was determined after differential centrifugation. All psoriatic enzymes showed much increased activities as compared to the normal stratum corneum enzymes. The highest activities of alkaline phosphatase from psoriatic scales could be detected in the nuclear fraction. The main activities of all other tested phosphatases and proteinases were present in the cytoplasmatic fraction. The subcellular distribution of the ribonucleases varied according to the pH value.

Separation of deoxyribonucleases (DNases) of normal human stratum corneum and psoriatic scales by micro-disc-electrophoresis.

Normal stratum corneum and psoriatic scales were homogenized and a differential centrifugation was performed. The DNase activity of the individual fractions was investigated by micro-dis-electrophoresis. At pH 5 only in the 600xg pellet and 105.000xg supernatant of normal keratin DNase activity could be observed. However, all psoriatic fractions showed distinct enzyme activities. At pH 7.4 little psoriatic DNase activity could only be demonstrated in the 105.000xg supernatant. Except from the 15.000xg pellet all fractions of normal stratum corneum displayed marked activities. In addition the 105.000xg supernatant showed two different DNase bands.

Separation and molecular weight estimation of phosphatases in psoriatic scales by disc-electrophoresis.

Disc-electrophoretic investigations of psoriatic scale homogenates (15000 x g supernatant) revealed several different phosphatase activities. At pH 5 either five different phosphatase active bands (substrates: p-nitrophenylphosphate, naphthylphosphate) or three different bands (substrate: glycerophosphate) could be obtained. At pH 7 only one band (substrate: p-nitrophenylphosphate) showed phosphatase activity. At pH 9.9 either two bands (substrate: p-nitrophenylphosphate) or three bands (substrate: glycerophosphate) could be demonstrated. the acid p-nitrophenylphosphatase activity of the psoriatic specific bands "9" and "10" could be distinguished by their different fluoride and tartrate susceptibility. Also the alkaline glycerophosphatase activities could be differentiated by distinct Ca and Mg susceptibility.