Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Antimycotic activity of fengycin C biosurfactant and its interaction with phosphatidylcholine model membranes.

Lipopeptide biosurfactants constitute one of the most promising groups of compounds for the treatment and prevention of fungal diseases in plants. Bacillus subtilis strain EA-CB0015 produces iturin A, fengycin C and surfactin and it has been proven useful for the treatment of black Sigatoka disease in banana plants, an important pathology caused by the fungus Mycosphaerella fijiensis (Morelet). We have found that B. subtilis EA-CB0015 cell free supernatants and purified fractions inhibit M. fijiensis cellular growth. The effect of the purified lipopeptides mentioned above on fungal growth has been also evaluated, observing that iturin A and fengycin C inhibit mycelial growth and ascospore germination, whereas surfactin is not effective. On the hypothesis that the antifungal action of the lipopeptides is associated to their incorporation into biological membranes, ultimately leading to membrane permeabilization, a detailed biophysical study on the interaction of a new isoform of fengycin C with model dipalmitoyphosphatidylcholine (DPPC) membranes has been carried out. Differential scanning calorimetry shows that fengycin C alters the thermotropic phase transitions of DPPC, and is laterally segregated in the fluid bilayer forming domains. Fluorescent probe polarization measurements show that fengycin C does not affect the hydrophobic interior of the membrane. This latter perturbation is concomitant with a strong dehydration of the polar region of DPPC, as shown by FTIR. Fengycin-rich domains, where the surrounding DPPC molecules are highly dehydrated, may well constitute sites of membrane permeabilization leading to a leaky target membrane. These results are a solid support to explain the membrane perturbing action of fengycin, which has been related to its antifungal activity.

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens.

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.

Sphingobacterium detergens sp. nov., a surfactant-producing bacterium isolated from soil.

A novel Gram-negative-staining strain, designated 6.2S(T), was isolated from a soil sample and identified as a biosurfactant producer. Its taxonomic position was investigated using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 °C, with 0-3% (w/v) NaCl, and at pH 7.0. Based on 16S rRNA gene sequence analysis, strain 6.2S(T) was found to be a member of the genus Sphingobacterium and was most closely related to four type species of the genus, showing sequence similarities of 96.8-98.9%. Partial chaperonin 60 (cpn60) gene sequence analysis was useful in resolving the phylogenetic relationships between strain 6.2S(T) and closely related taxa, with similarities ranging from 85.5% (with Sphingobacterium thalpophilum DSM 11723(T)) to 90.3% (with Sphingobacterium canadense CR11(T) and Sphingobacterium multivorum JCM 21156(T)). The results of DNA-DNA hybridization experiments between the novel strain and its closest relatives gave a DNA-DNA relatedness value of less than 70%, and consequently confirmed that this new strain did not belong to a previously described species of the genus Sphingobacterium. The major fatty acids were summed feature 3 (iso-C(15:0) 2 OH and/or C(16:1)ω7c); iso-C(15:0); iso-C(17:0) 3-OH and C(16:0). The G+C content of the genomic DNA was 40.0 mol%. According to its phenotypic and genotypic characteristics and the phylogenetic data, strain 6.2S(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium detergens sp. nov. is proposed. The type strain is 6.2S(T) ( = CECT 7938(T) = LMG 26465(T)).

Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function.

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.