Epidermal keratin 5 expression and distribution is under dermal influence.

Human skin melanin pigmentation is regulated by systemic and local factors. According to the type of melanin produced by melanocytes, the transfer and degradation of melanosomes differ, thus accounting for most variations between ethnicities. We made the surprising observation that in a drastically changed environment, white and black phenotypes are reversible since Caucasian skin grafted onto nude mice can become black with all black phenotypic characteristics. Black xenografts differed essentially from other grafts by the levels of epidermal FGF-2 and keratin 5. In vitro analysis confirmed that FGF-2 directly regulates keratin 5. Interestingly, this phenomenon may be involved in human pathology. Keratin 5 mutations in Dowling-Degos Disease (DDD) have already been associated with the pheomelanosome-eumelanosome transition. In a DDD patient, keratin 5 was expressed in the basal and spinous layers, as observed in black xenografts. Furthermore, in a common age-related hyperpigmentation disorder like senile lentigo (SL), keratin 5 distribution is also altered. In conclusion, modulation of keratin 5 expression and distribution either due to mutations or factors may account for the development of pigmentary disorders.

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster Palinurus gilchristi.

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 x 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 x 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin-oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0-1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium-hemocyanin interaction, three other species belonging to Palinura.

Melanoma versus dysplastic naevi: microtopographic skin study with noninvasive method.

Malignant melanoma (MM) is a cutaneous tumour that originates from the epidermal melanocytes, and whose prognosis is strongly determined by tumour vertical thickness. ABCDE system is currently used for the clinical diagnosis of MM: lesion asymmetry, border irregularity, change or variegation in colour, diameter > 6mm and rapid evolution. Different methods have been used to improve the diagnosis of MM, but the results have not been convincing. Our work investigates the differences between MM and atypical naevi through skin surface analysis, using scanning electron microscopy (SEM): in fact ABCDE classical criteria are often insufficient for differential diagnosis of melanoma. In our study, we analysed 15 malignant melanomas surfaces from 15 patients and of 15 atypical naevi from 10 patients. On histological examination five superficial spreading melanoma (SSM) and 10 nodular melanoma (NMM) were found. Inclusion criteria used for selection were clinical diagnosis of SSM or clinical doubt between MM and atypical naevi (dysplastic naevi). Superficial skin texture was analysed using the silicone replica technique. The replica material is a polyvinylsiloxane derivative for dental application. Our results show that the melanoma has a very irregular surface, with a marked reduction or disappearance of cutaneous furrows, and formation of new lines with varying direction. In conclusion, the method above based on skin surface analysis, may be of help in diagnosis of early stage MM and in differentiating from atypical naevi.

The hemocyanin of the shamefaced crab Calappa granulata: structural-functional characterization.

Arthropod hemocyanins (Hcs) transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Calappa granulata Hc is found as a mixture of dodecamers (95%) and hexamers (5%). Removal of calcium ions and alkaline pH induce an incomplete partially reversible dissociation of dodecameric Hc. Two-dimensional electrophoretic pattern of dissociated Hc indicated a large heterogeneity in Hc subunit: most differences are likely to be explained by post-translational modifications. Dodecameric Hc showed a large Bohr effect (Deltalog P50/DeltapH = -0.95) and a normal cooperativity (h50 values = 2.7 +/- 0.2) in the presence of 10 mM CaCl2. The hexameric molecule displayed lower Bohr effect and cooperativity than the dodecamer. Lactate effect on the oxygen affinity (Deltalog P50 = 0.55) and the increase of lactate concentrations in animals kept in emersion were related to the increased oxygen requirements that occur during hypoxia in vivo. Calcium affects oxygen affinity only at high concentrations: this Hc appeared to lack the calcium high-affinity binding sites found in other species. The effect of temperature on both oxygen affinity and cooperativity was measured in the absence and presence of 10 mM lactate, allowing calculation of the exothermic contribution of lactate binding (DeltaH = -25 kJ mol(-1)).

Two sites for GTP binding in cathodic haemoglobins from Anguilliformes.

Cathodic haemoglobins of four species of anguilliform fish were characterized from a functional point of view, with special regard to the interaction with their physiological effectors. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of saturating amounts of chloride. The results indicated that the cathodic haemoglobin of three species (Anguilla anguilla, Conger conger and Muraena helena) do have two sites for GTP-binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for the second GTP-binding site. The cathodic haemoglobin of Gymnothorax unicolor, which showed lower GTP sensitivity than the other haemoglobins examined, displayed only one GTP-binding site. The presence of an additional phosphate-binding site is not exceptional, although the way haemoglobin interacts with the two organic phosphate molecules may differ among species. This property may provide an auxiliary means of haemoglobin modulation for species that inhabit environments where oxygen availability is highly variable and haemoglobin-oxygen affinity needs to be modulated to different extents in order to satisfy physiological oxygen requirements.

Functional and structural characterization of the myoglobin from the polychaete Ophelia bicornis.

The myoglobin of the polychaete annelid Ophelia bicornis was isolated, purified to homogeneity and characterized. The primary structure, obtained from cDNA and protein sequencing, consists of 139 amino acid residues. The alignment with other globin sequences showed that O. bicornis myoglobin misses the pre-A helix and the first six residues of the A helix. The presence of a PheB10-GlnE7 haem distal residue pair is in agreement with the measured oxygen affinity (P50=0.85 mmHg; 1 mmHg=0.133 kPa) and the only slightly higher autoxidation rate constant (0.28 h(-1)) with respect to that of the sperm whale myoglobin mutant E7 His-->Gln (0.21 h(-1)) and to elephant myoglobin (0.1 h(-1)). Oxygen-binding co-operativity was found to be absent under all the examined experimental conditions. The resistance of O. bicornis myoglobin towards autoxidation seems to confirm the important role of part of the A helix in the stability of the globin. The higher pKa of the acid-alkaline ferric transition of O. bicornis with respect to Asian elephant myoglobin, as well as the higher absorbance ratio of its ferric form to the oxy form measured in the Soret region (gammamet/gammaoxy) with respect to that of the African elephant myoglobin, suggested a stronger interaction between the distal glutamine and the water molecule at the sixth co-ordinate position.