P53 in skin cancer: From a master player to a privileged target for prevention and therapy.

The increasing incidence of skin cancer (SC) is a global health concern. The commonly reported side effects and resistance mechanisms have imposed the pursuit for new therapeutic alternatives. Moreover, additional preventive strategies should be adopted to strengthen prevention and reduce the rising number of newly SC cases. This review provides relevant insights on the role of p53 tumour suppressor protein in melanoma and non-melanoma skin carcinogenesis, also highlighting the therapeutic potential of p53-targeting drugs against SC. In fact, several evidences are provided demonstrating the encouraging outcomes achieved with p53-activating drugs, alone and in combination with currently available therapies in SC. Another pertinent perspective falls on targeting p53 mutations, as molecular signatures in premature phases of photocarcinogenesis, in future SC preventive approaches. Overall, this review affords a critical and timely discussion of relevant issues related to SC prevention and therapy. Importantly, it paves the way to future studies that may boost the clinical translation of p53-activating agents, making them new effective alternatives in precision medicine of SC therapy and prevention.

Efeito da suplementação de suínos em terminação com diferentes associações entre minerais sobre o desempenho, as características de carcaça e a viabilidade econômica / Effect of supplementation of finishing swines with different associations between minerals on performance, carcass characteristics and economic viability

Objetivou-se, com este estudo, verificar a influência da suplementação com diferentes associações entre minerais orgânicos sobre o desempenho e as características de carcaça, bem como avaliar sua viabilidade econômica, para suínos em terminação. Foram utilizados 88 machos castrados, divididos entre quatro tratamentos: controle com dieta basal e suplementação com CrFe, MgSe e CrFeMgSe, durante os 28 dias que antecederam o abate. O delineamento experimental foi em blocos ao acaso, com parcela experimental representada por dois animais. Os animais suplementados com CrFeMgSe apresentaram piores índices de peso final, ganho de peso diário e consumo durante todo o período, com consequente redução no peso de carcaça, menor profundidade de lombo, área de olho-de-lombo e menor quantidade de carne magra na carcaça. Houve um aumento no custo da dieta para os grupos CrFe, MgSe e CrFeMgSe, respectivamente. O uso de cromo mais ferro e magnésio mais selênio, associados dois a dois, não altera os parâmetros de desempenho e as características de carcaça. A associação dos quatro minerais, no entanto, promove piora nessas características e aumenta o custo da dieta.(AU)

This study aimed to verify the influence of supplementation with different associations between organic minerals on performance, carcass characteristics and economic viability for finishing swine. 88 castrated males were divided in four treatments: control with basal diet and supplementation with CrFe, MgSe and CrFeMgSe during the 28 days prior to slaughter. The experimental design was randomized blocks with an experimental plot represented by two animals. The animals supplemented with CrFeMgSe had worse final weight, daily weight gain and consumption throughout the period, with consequent reduction in carcass weight, lower loin depth, loin eye area and amount of lean meat. There was an increase in the effective operational cost of diet with CrFe, MgSe and CrFeMgSe groups, respectively. The use of chromium plus iron and magnesium plus selenium, associated two by two, did not alter the performance parameters and carcass. However, the four minerals association promote a worsening in these characteristics and increase the cost of production.(AU)

One-year follow-up of the immune profile in serum and selected sites of generalized and localized aggressive periodontitis.

BACKGROUND: The local and systemic immunological profiles of important inflammatory mediators in the localized (LAgP) and generalized (GAgP) forms of aggressive periodontitis are still unknown, as well as the effect of periodontal therapy on these parameters. The aim of this prospective study was to evaluate clinical and immune responses of patients with AgP undergoing nonsurgical treatment. MATERIAL AND METHODS: Eighteen patients with GAgP, 10 with LAgP and 10 healthy participants were included in this study. AgP participants were submitted to scaling and root planing plus systemic antibiotics (amoxicillin and metronidazole). At baseline and 1-year follow-up were measured clinical parameters, such as probing depth [PD] and clinical attachment loss [CAL], and the levels of 10 immunological mediators (GM-CSF, M-CSF, MCP-1, ICAM-1, CXCL8, IL-1ß, TNF-α, IL-17, IL-4, and IL-10) in the gingival crevicular fluid (GCF) of selected sites [AgP forms: PD ≥ 6 mm or the deepest, bleeding on probing (BoP) and bone loss measured by periapical radiography; healthy individuals: PD ≤ 3 mm, no BoP, no bone loss] and serum. RESULTS: After periodontal treatment both forms of AgP presented a significant reduction of PD and CAL, an increase of GM-CSF, ICAM-1, MCP-1, TNF-α, IL-17, IL-4, and IL-10 in the GCF, as well as of GM-CSF and IL-4 in the serum, and a reduction in the serum concentration of IL-1ß. Serum levels of M-CSF, ICAM-1, and MCP-1 remained significantly below those found in healthy individuals in both forms of AgP even after therapy. An increase in the systemic or local levels of MCP-1, ICAM-1 and the anti-inflammatory profile (IL-4, IL-10) was correlated with an improvement in clinical parameters of LAgP patients. Also, a local reduction of IL-1ß levels in both forms of AgP was correlated with an increase in the clinical attachment gain. CONCLUSION: Nonsurgical periodontal therapy was successful in improving clinical parameters and modulating the immune response in both forms of AgP. However, this therapeutic approach does not seem to affect the deficient level of important serum mediators involved in mechanisms of cell transmigration.

Immunological and microbiological periodontal profiles in isolated growth hormone deficiency.

BACKGROUND: Growth hormone (GH) has been identified as an important regulator of the immune response. We have previously shown that adults with isolated GH deficiency (IGHD) due to a mutation in the GH releasing hormone receptor (GHRHR) gene, have a greater chance of having periodontitis. However, the interaction of GH with periodontal tissues is still unknown, and this population has emerged as a unique model to investigate this issue. Therefore, we evaluated the microbiological and immunological periodontal profiles of such individuals. METHODS: Nineteen IGHD and 19 controls matched by age, sex, diabetes, and smoking status, were enrolled in this case-control study. Periodontal clinical parameters (probing depth [PD] and clinical attachment loss [AL]) were measured at six sites per tooth. Immune mediators (C-reactive protein, matrix metalloproteinase [MMP]-8, MMP-9, interleukin [IL]-1α, IL-6, IL-8, tumor necrosis factor [TNF]-α, adiponectin, and leptin) were analyzed by enzyme-linked immunosorbent assay (ELISA) in the gingival crevicular fluid (GCF) in four non-adjacent sites for each participant (two with PD ≤3 mm [shallow sites] and two with PD ≥7 mm or the worst PD found in the mouth [deep sites]). Bacterial quantification (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) of subgingival biofilm samples collected from these same sites was performed by quantitative real-time polymerase chain reaction (qPCR). RESULTS: IGHD individuals presented higher values of PD and AL, and increased levels of CRP, IL-8, MMP-8, and adiponectin in the GCF. Bacterial quantification did not identify differences between the two groups. CONCLUSION: IGHD alters the local immune response in periodontal pockets leading to greater attachment loss, and GH stands out as an important hormone to be evaluated in the pathogenesis of periodontitis.

E. coli promotes human Vγ9Vδ2 T cell transition from cytokine-producing bactericidal effectors to professional phagocytic killers in a TCR-dependent manner.

γδT cells provide immune-surveillance and host defense against infection and cancer. Surprisingly, functional details of γδT cell antimicrobial immunity to infection remain largely unexplored. Limited data suggests that γδT cells can phagocytose particles and act as professional antigen-presenting cells (pAPC). These potential functions, however, remain controversial. To better understand γδT cell-bacterial interactions, an ex vivo co-culture model of human peripheral blood mononuclear cell (PBMC) responses to Escherichia coli was employed. Vγ9Vδ2 cells underwent rapid T cell receptor (TCR)-dependent proliferation and functional transition from cytotoxic, inflammatory cytokine immunity, to cell expansion with diminished cytokine but increased costimulatory molecule expression, and capacity for professional phagocytosis. Phagocytosis was augmented by IgG opsonization, and inhibited by TCR-blockade, suggesting a licensing interaction involving the TCR and FcγR. Vγ9Vδ2 cells displayed potent cytotoxicity through TCR-dependent and independent mechanisms. We conclude that γδT cells transition from early inflammatory cytotoxic killers to myeloid-like APC in response to infectious stimuli.

Validity and reliability of a pictorial instrument for assessing perceived motor competence in Portuguese children.

BACKGROUND: It is important to assess young children's perceived Fundamental Movement Skill (FMS) competence in order to examine the role of perceived FMS competence in motivation toward physical activity. Children's perceptions of motor competence may vary according to the culture/country of origin; therefore, it is also important to measure perceptions in different cultural contexts. The purpose was to assess the face validity, internal consistency, test-retest reliability and construct validity of the 12 FMS items in the Pictorial Scale for Perceived Movement Skill Competence for Young Children (PMSC) in a Portuguese sample. METHODS: Two hundred one Portuguese children (girls, n = 112), 5 to 10 years of age (7.6 ± 1.4), participated. All children completed the PMSC once. Ordinal alpha assessed internal consistency. A random subsamples (n = 47) were reassessed one week later to determine test-retest reliability with Bland-Altman method. Children were asked questions after the second administration to determine face validity. Construct validity was assessed on the whole sample with a Bayesian Structural Equation Modelling (BSEM) approach. The hypothesized theoretical model used the 12 items and two hypothesized factors: object control and locomotor skills. RESULTS: The majority of children correctly identified the skills and could understand most of the pictures. Test-retest reliability analysis was good, with an agreement ration between 0.99 and 1.02. Ordinal alpha values ranged from acceptable (object control 0.73, locomotor 0.68) to good (all FMS 0.81). The hypothesized BSEM model had an adequate fit. CONCLUSIONS: The PMSC can be used to investigate perceptions of children's FMS competence. This instrument can also be satisfactorily used among Portuguese children.

Gingival crevicular fluid levels of protease-activated receptors type 1 and type 2 in diabetic patients with periodontitis.

BACKGROUND AND OBJECTIVE: Protease activated receptor type 1 (PAR1 ) seems to play a role in periodontal repair, while PAR2 is associated with periodontal inflammation. As diabetes is a known risk factor for periodontal disease, the aim of this study was to evaluate the influence of type 2 diabetes on PAR1 and PAR2 mRNA expression in the gingival crevicular fluid of patients with chronic periodontitis before and after non-surgical periodontal treatment. MATERIAL AND METHODS: Gingival crevicular fluid samples and clinical parameters consisting of measuring probing depth, clinical attachment level, bleeding on probing and plaque index were collected from systemically healthy patients and patients with type 2 diabetes and chronic periodontitis, at baseline and after non-surgical periodontal therapy. PAR1 and PAR2 , as well as the presence of the proteases RgpB gingipain and neutrophil proteinase-3 were assessed by quantitative polymerase chain reaction in the gingival crevicular fluid. RESULTS: The periodontal clinical parameters significantly improved after periodontal therapy (p < 0.01). Diabetes led to increased expression of PAR1 in gingival crevicular fluid, and in the presence of chronic periodontitis, it significantly decreased the expression of PAR1 and PAR2 (p < 0.05). Moreover, non-surgical periodontal treatment in diabetics resulted in increased expression of PAR1 and PAR2 (p < 0.05), and decreased expression of RgpB gingipain and proteinase-3 (p < 0.05). CONCLUSION: The present data demonstrated that diabetes was associated with an altered expression of PAR1 and PAR2 in the gingival crevicular fluid cells of subjects with chronic periodontitis. Future studies are necessary to elucidate the effects of PAR1 upregulation in periodontally healthy sites and PAR2 downregulation in chronic periodontitis sites on the increased susceptibility and severity of periodontitis in diabetes.

Hydrogen peroxide-induced secondary necrosis in conidia of Aspergillus fumigatus.

Aspergillus fumigatus conidia have been linked to severe aspergillosis in immunocompromised patients. Recently, the cytotoxic effect of secondary metabolites from A. fumigatus conidia was reported. In the present work, a methodology used to detect cell death markers in fungal hyphae was adapted to study conidia cell death. Additionally, the mechanism of H2O2-induced cell death was studied in A. fumigatus conidia for the first time. Data presented in this work reveal that the H2O2-induced conidial cell death was associated with a marked increase of TUNEL- and PI-positive cells. It is therefore suggested that conidia cell death occurs in a dose-dependent manner through a secondary necrosis mechanism. The knowledge of conidia cell death machinery may provide insights into the molecular mechanism of conidia-mediated toxicity to the respiratory tract and may pave the way for improved therapeutic approaches against A. fumigatus conidia-mediated diseases.

Phlebotomine sand flies (Diptera: Psychodidae) and Leishmania infection in Gafanhoto Park, Divinópolis, Brazil.

The potential of Gafanhoto Park as an American cutaneous leishmaniasis (ACL) focus was evaluated by examination of sand fly vectors of the Leishmania parasite. This forest remnant is located in a periurban area of Divin6polis, Brazil, where autochthonous cases of ACL have been reported. Sand fly populations were monitored over a 2-yr period (2006-2008) by using light traps (HP and Shannon). During systematic collections with HP traps, 824 specimens in total (342 males and 482 females) of 21 species were captured. Most prevalent species were as follows: Brumptomyia brumpti (Larrouse), Lutzomyia aragaoi (Costa Lima), Lutzomyia lutziana (Costa Lima), Lutzomyia sordellii (Shannon & Del Ponte), and Lutzomyia whitmani (Antunes & Coutinho). Using Shannon traps, 257 specimens representing 15 species were collected (159 females and 98 males), with a high prevalence of L. whitmani and Lutzomyia neivai (Pinto), both vectors of Leishmania braziliensis (Vianna). To ascertain the level of natural infection, a sample of females captured in Shannon traps was assayed for the presence of Leishmania by using polymerase chain reaction-restriction fragment length polymorphism, where 39% of insects were positive. The most infected species was L. whitmani (29 sand flies; 18.2%), followed by L. neivai (21; 13.2%), Lutzomyia christenseni (Young & Duncan) (five; 3.1%), Lutzomyia pessoai (Coutinho & Barreto) (three; 1.9%), L. aragaoi (one; 0.6%), Lutzomyia fischeri (Pinto) (one; 0.6%), Lutzomyia lenti (Mangabeira) (one; 0.6%), L. lutziana (one; 0.6%), and Lutzomyia monticula (Costa Lima) (one; 0.6%). The finding of potential and incriminated vectors naturally infected with Leishmania reinforces the need of epidemiologic surveillance in the area.

Selective activation of protein kinase C-delta and -epsilon by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U).

6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC-alpha and -betaI), novel (nPKC-delta and -epsilon) and atypical (aPKC-zeta), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC, coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs. The isoform-selectivity of coleon U for nPKC-delta and -epsilon was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase- and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC, recognised as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC-delta and -epsilon activation, it represents a promising compound for evaluation as an anti-cancer drug.

Evaluation of delayed neuronal and axonal damage secondary to moderate and severe traumatic brain injury using quantitative MR imaging techniques.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) is a classic model of monophasic neuronal and axonal injury, in which tissue damage mainly occurs at the moment of trauma. There is some evidence of delayed progression of the neuronal and axonal loss. Our purpose was to test the hypothesis that quantitative MR imaging techniques can estimate the biologic changes secondary to delayed neuronal and axonal loss after TBI. MATERIALS AND METHODS: Nine patients (age, 11-28 years; 5 male) who sustained a moderate or severe TBI were evaluated at a mean of 3.1 years after trauma. We applied the following techniques: bicaudate (BCR) and bifrontal (BFR) ventricle-to-brain ratios; T2 relaxometry; magnetization transfer ratio (MTR); apparent diffusion coefficient (ADC); and proton spectroscopy, by using an N-acetylaspartate/creatine (NAA/Cr) ratio measured in normal-appearing white matter (NAWM) and the corpus callosum (CC). The results were compared with those of a control group. RESULTS: BCR and BFR mean values were significantly increased (P < or = .05) in patients due to secondary subcortical atrophy; increased T2 relaxation time was observed in the NAWM and CC, reflecting an increase in water concentration secondary to axonal loss. Increased ADC mean values and reduced MTR mean values were found in the NAWM and CC, showing damage in the myelinated axonal fibers; and decreased NAA/Cr ratio mean values were found in the CC, indicating axonal loss. CONCLUSIONS: These quantitative MR imaging techniques could noninvasively demonstrate the neuronal and axonal damage in the NAWM and CC of human brains, secondary to moderate or severe TBI.

Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.

Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear "ferroxidase centre" in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redox-inactive Zn(II). The results for Fe-rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex-treated. The X-ray absorption near-edge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal-ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex-treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries.

Mitochondria-dependent apoptosis in yeast.

Mitochondrial involvement in yeast apoptosis is probably the most unifying feature in the field. Reports proposing a role for mitochondria in yeast apoptosis present evidence ranging from the simple observation of ROS accumulation in the cell to the identification of mitochondrial proteins mediating cell death. Although yeast is unarguably a simple model it reveals an elaborate regulation of the death process involving distinct proteins and most likely different pathways, depending on the insult, growth conditions and cell metabolism. This complexity may be due to the interplay between the death pathways and the major signalling routes in the cell, contributing to a whole integrated response. The elucidation of these pathways in yeast has been a valuable help in understanding the intricate mechanisms of cell death in higher eukaryotes, and of severe human diseases associated with mitochondria-dependent apoptosis. In addition, the absence of obvious orthologues of mammalian apoptotic regulators, namely of the Bcl-2 family, favours the use of yeast to assess the function of such proteins. In conclusion, yeast with its distinctive ability to survive without respiration-competent mitochondria is a powerful model to study the involvement of mitochondria and mitochondria interacting proteins in cell death.

Body composition in patients with chronic obstructive pulmonary disease: which method to use in clinical practice?

The objective of the present study was to compare anthropometry with bioelectrical impedance (BIA) in relation to densitometry (dual-energy X-ray absorptiometry; DEXA) as methods of nutritional assessment and body composition in out-patients with chronic pulmonary obstructive disease (COPD). We conducted a cross-sectional clinical study with sixty-one patients with COPD (forty-two men and nineteen women), mean age of 66.5 (sd 7.9) years and forced expiratory volume in 1 s of 1.3 (sd 0.6) litres (52.2 (sd 19.8) % predicted), referred to the Pulmonary Rehabilitation Center. The patients were evaluated regarding nutrition status and body composition as determined by anthropometry, BIA and DEXA. In the results, 34.4 % showed mild obstruction, 31.2 %, moderate and 34.4 %, severe obstruction. According to the BMI (mean 24.5 (sd 4.5) kg/m2), 45.9 % of the patients exhibited normal weight, while 27.9 % were underweight and 26.2 % were obese. Related to fat-free mass (FFM), anthropometry and BIA compared with DEXA presented high correlations (r 0.96 and 0.95 respectively; P < 0.001) and high reliability between the methods (alpha 0.98; P < 0.001). Agreement analysis between the methods shows that anthropometry overestimates (0.62 (sd of the difference 2.89) kg) while BIA underestimates FFM (0.61 (sd of the difference 2.82) kg) compared with DEXA. We concluded that according to the nutritional diagnosis, half of our population of patients with COPD showed normal weight, while the other half comprised equal parts obese and underweight patients. Body composition estimated by BIA and anthropometry presented good reliability and correlation with DEXA; the three methods presented satisfactory clinical accuracy despite the great disparity of the limits of agreement.

Variations in BOD, algal biomass and organic matter biodegradation constants in a wind-mixed tropical facultative waste stabilization pond.

This study considered the impact of wind mixing on the efficiency of BOD removal and the first order biodegradation constant for organic matter in a primary facultative pond. Wind speeds of 1-4 m/s blowing from the effluent end of the pond towards the influent created surface-water flows of up to 0.94 m/s as determined by orange and coconut drogues moving in the opposite direction to the bulk hydraulic flow of 0.217 m/s. This was sufficient to cause mixing of the water column resulting in loss of stratification in terms of chlorophyll a, temperature and dissolved oxygen. BOD and chlorophyll a concentrations were spatially and temporally homogeneous throughout this large pond. BOD removal efficiency was only 50.30% as opposed to a projected value of 79% despite an acceptable surface organic loading of 350 kgBOD5/ha/d and an actual k value for BOD removal using influent sewage samples of 0.29 d-1 close to the projected value of 0.30 d-1. It would seem that wind mixing reduced pond efficiency by destroying stratification and thus reducing the microbial activity necessary to consume organic material. Mixing also increased the mean chlorophyll a concentration compared to stratified facultative ponds receiving similar loads and non-motile algae dominated the water column.

Determination of the sedimentation constants for total suspended solids and the algal component in a full-scale primary facultative pond operating at high wind velocities under tropical conditions.

This study evaluated the amount, distribution and sedimentation constant of solids in a full-scale primary facultative pond operating mostly under high wind conditions and the contribution made by the algal biomass. Solids deposition rates were measured using sedimentation traps placed in the inlet and outlet zones of the pond. Most sludge accumulation occurred, not surprisingly, in the inlet zone A1 with a sludge volume of 9072.m3 accumulating over an operating time of approximately 3 years. However, sludge deposition within this zone was uneven and affected by wind action. Mean proportionality constant (K) values for solids sedimentation were 3.02 and 5.70 for depths of 50 cm and 100 cm respectively for A1. In contrast in zone A3, (the outlet zone), reduced K values of 1.38 and 3.22 were obtained for depths of 50 cm and 100 cm respectively. The algal sedimentation constant varied from 0.8 d(-1) in zone A1 to 0.02 d(-1) in A3. These data suggest that in this large facultative pond the wind, blowing predominantly from the direction of the outlets towards the pond inlets, had a greater influence on solids deposition than the bulk hydraulic flow and also kept the pond completely mixed for most of the time.

Differential activation by daphnetoxin and mezerein of PKC-isotypes alpha, beta I, delta and zeta.

Daphnetoxin, a mezerein derivative, was isolated from the stem bark of Daphne gnidium. Mezerein is a PKC activator that exhibits antileukemic properties. However, daphnetoxin and its analogue 12-hydroxydaphnetoxin were described as being devoid of this effect. In the present study daphnetoxin and mezerein were compared as PKC activators on classical (alpha and beta I), novel (delta) and atypical (zeta) isoforms, using an alternative in vivo yeast phenotypic assay. The aim was to clarify if daphnetoxin is a PKC activator and if the differences between the antiproliferative effect of mezerein and of its analogue daphnetoxin may be ascribed to differences on their potency or selectivity as PKC activators. Yeast samples expressing each of the mammalian PKC isoforms tested were incubated with daphnetoxin or mezerein. Growth inhibition caused by these drugs was assumed to be due to PKC activation since it did not occur when expression was not induced. Mezerein inhibited the growth of yeast expressing PKC alpha (IC(50) = 1190 +/- 237 nM; n = 20), PKC beta I (IC(50) = 908 +/- 46 nM; n = 20), and PKC delta (IC(50) = 141 +/- 25 nM; n = 20) but not of yeast expressing PKC zeta. Daphnetoxin also inhibited the growth of yeast expressing isoforms alpha, beta I and delta, being more potent than mezerein on PKC alpha (IC(50) = 536 +/- 183 nM; n = 20; P < 0.05), as potent as mezerein on PKC beta I (IC(50) = 902 +/- 129 nM; n = 20) and less potent than mezerein upon PKC delta (IC(50) = 3370 +/- 492 nM; n = 20; P < 0.05). These results show that daphnetoxin is a potent PKC activator but with a selectivity different from that of mezerein. It is suggested that the lack of antileukemic and antiproliferative effects of daphnetoxin may be due to its lower potency to activate PKC delta.

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.