Laboratory markers of melanoma progression

Extracellular tumour markers may have potential role in the follow-up of patients with malignant melanoma, in therapy monitoring and in prediction of prognosis. In our article circulating tumour markers in melanoma (melanoma inhibitory activity, lipid bound sialic acid, neuron specific enolase, TA90 immune complex, S-100B protein, 5-S-cysteinyldopa, tyrosinase, cytokines, metalloproteinases, LDH) were reviewed. Among laboratory melanoma markers the S-100B protein is the most investigated. S-100B protein has high specificity, appropriate sensitivity and proved to be significant prognostic factor independent from stages. High serum values are associated with shorter survival. However, before S-100B monitoring immunohistochemistry for the detection of S-100B is required. In the case of malignant melanomas with low expression serum S-100B monitoring may not be sensitive enough to follow disease progression. Although the serum concentration of 5-S-cysteinyldopa did not prove to be independent prognostic factor in our previous studies comprising the highest patient number in the literature, the marker was suggested for therapy monitoring. The survival analysis indicated that the elevated 5-S-cysteinyldopa level predicts shorter survival. In spite of the calculated low correlation between the two markers, parallel elevation of S-100B protein and 5-S-cysteinyldopa indicated shorter survival. On the basis of the literature LDH is the most appropriate tumour marker in stage IV to predict prognosis, but its sensitivity and specificity could not achieve that of S-100B protein. S-100B and LDH proved to be similarly reliable in respect to the clinical outcome. Determination of serum concentration of MIA and tyrosinase are also reliable markers in malignant melanoma. The other investigated markers are not well known yet or do not provide useful information to the clinicians.     

Naevus pigmentosus and melanoma

Clinical observations and histological findings support the relationship between pigmented naevi and melanoma. The author describes the morphological characteristics of congenital, acquired and atypical naevi in relation to the appearance of melanoma. On the basis of clinical observations, in harmony with other investigators, the author advises patients to perform self examination and to undergo regular survey of numerous atypical naevi by a dermatologist. In fact, any of the atypical pigmented naevi present in high number may be a precursor lesion. Patients with such lesions are at higher risk of melanoma development.     

Comparison of traditional and molecular typing methods of Escherichia coli O157

An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.     

The Arabidopsis anaphase-promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit

In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.     

Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings

The effect of some more or less harmful compounds like Cd, Pb, Ni, Ti salts and DCMU at low concentrations on the development of chloroplasts in maize and bean seedlings was investigated. Chlorophyll content, chlorophyll a/b ratio, photosynthetic activity (14CO2 fixation), chlorophyll-protein composition of thylakoid membranes, fluorescence spectra of chloroplasts, fluorescence induction parameters of leaves and electron microscopic structure of maize and bean chloroplasts as well as growth parameters were studied. Stimulation of chlorophyll synthesis and photosynthetic activity was observed at different intervals during all of the treatments, while chlorophyll a/b ratios and fluorescence properties of leaves or chloroplasts did not change considerably except in DCMU treated plants. Heavy metal treatments increased the amount of photosystem I and light-harvesting complex II, while decreased amount of photosystem I and higher amount of light-harvesting complex II was found in DCMU treated thylakoids. Electron microscopy showed only sligth differences in the morphology of chloroplast lamellar system (mostly in DCMU treated plants), while the status of the plasmalemma and tonoplast seemed to be altered as a result of certain metal treatments. Results showed the expression of a cytokinin-like effect on the development of chloroplasts. It is assumed, that these low-dose stressors generate non-specific alarm reactions in plants, which may involve changes of the hormonal balance.     

C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins

MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells. It has been reported that the trafficking of MRP2 is modulated by PKC. Adjacent to the C-terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser(1542)). Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, beta2-syntrophin, and SAP-97). The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied. We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas beta2-syntrophin exhibited binding only to ABCA1. The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50. Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2.     

Activation parameters of the blue shift (Shibata shift) subsequent to protochlorophyllide phototransformation

The Shibata shift was analyzed in flash irradiated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates in the pressure range of 0.1 to 500 MPa, at temperatures of 20, 30 and 40 degrees C. The kinetics of the blue shift (called Shibata shift in case of intact leaves) was followed by repeated recording of fluorescence emission spectra after phototransformation. At 20 degrees C, above 100 MPa, the blue shift slowed down remarkably. Two components of the blue shift could be distinguished, one was pressure-dependent and the other was almost pressure-independent. The pressure-independent component can be associated with minor conformational changes of the NADPH:protochlorophyllide oxidoreductase (POR) enzyme, followed by molecular movements of the newly formed chlorophyllide molecules. The calculated activation volume of the pressure-dependent component was 43+/-11 cm(3) mol(-1) at 20 degrees C. This value reflects major molecular reorganizations in the lipid system of the membrane and in the chlorophyllide-protein complexes, and corresponds to changes of the tertiary structure of proteins which can proceed directly or indirectly via structural changes of the membrane lipids. The process was inhibited by 300 and 400 MPa at 30 and 40 degrees C, respectively. The activation volume reduced to 35+/-1.5 cm(3) mol(-1) at 40 degrees C. The decrease of the activation volume with increasing temperature indicates that the blue shift requires loosened lipid structures. The activation energy of the blue shift (measured between 10 and 40 degrees C at atmospheric pressure) was 100+/-20 kJ/mol, indicating that the structural change involves rearrangement of strong molecular interactions.