The urological referral process from primary health care. Reflections on referral criteria.

OBJECTIVES: The flow of patients between Primary Care (PC) and Specialized care (SC) is a common process. It carries many implications for the patient, physician and health system. In Urology, only benign prostatic hyperplasia (BPH) has referral criteria. Urinary incontinence, prostate cancer (PCa), and urological ultrasound, are in the process. The aim of this paper is to communicate, with critical analysis, the characteristics of the information recorded in the referral visit (clinical reasons / rationale) and the effectiveness for urology consultation. METHODS: Observational, descriptive and quantitative study of the referral visits made between PC/SC (Urology) in the health care area of our hospital (December 2010-September 2012). We studied: Referral Visit Database (RVD), consultation document, HORUS system, and specific referral visit survey questionnaire. RESULTS. Referral visits account for 67.89% (all first consultations), 14.79% of the total number of visits. 78% were male (mean age 53 y.o). 11.84% recorded reason for consultation (98% in referral document) with normal priority (94.67%). 34% of them were for BPH. HORUS is not exploited for the referral visit. 40% start the diagnostic process with insufficient exams. 18.1% are listed as closed process / completed. Patient satisfaction was evaluated (20%). Key points in the improvement are: improve referral visit reason for consultations, to know patient's expectations, and to develop protocols (guidelines, and/or referral criteria). CONCLUSIONS. The referral process is complex. The computer system does not include the referral reason for consultation. Institutional agreement between PC/SC Urology must be reached to ensure uniformity in the implementation and support.

Buthionine sulfoximine diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.

Buthionine sulfoximine (BSO) is a specific inhibitor of γ-glutamylcysteine synthetase, thus blocking the synthesis of glutathione (GSH). It is known that this makes that BSO affects melanin synthesis because of the role of thiols in melanogenesis. However, BSO may also react with the intermediate oxidation products of melanogenesis, a possibility that has not been investigated from the initial steps of the pathway. We created in vitro conditions simulating eumelanogenesis (oxidation of L-DOPA in the absence of GSH) and pheomelanogenesis (oxidation of L-DOPA in the presence of GSH) under presence or absence of BSO. BSO made that eumelanogenesis results in pigments more soluble and less resistant to degradation by hydrogen peroxide than pigments obtained without BSO. A similar but less marked effect was observed for pheomelanogenesis only at subsaturating concentrations of GSH. These results suggest that BSO diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.

Raman spectroscopy as a non-invasive technique for the quantification of melanins in feathers and hairs.

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non-invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4-amino-3-hydroxyphenylalanine, 4-AHP) and eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA) as measured by HPLC in feathers of red-legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4-AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.

Vibrational characterization of pheomelanin and trichochrome F by Raman spectroscopy.

We characterize for the first time the vibrational state of natural pheomelanin using Raman spectroscopy and model pigment synthesized from 5-S-cysteinyldopa. The shape of the Raman spectrum was very different from that of eumelanin. Four Raman bands were visible in the 500-2000 cm(-1) wavenumber region about 500, 1150, 1490 and 2000 cm(-1), which we assigned to the out-of-plane deformation and the stretching vibration of the phenyl rings, to the stretching vibration of C-N bonds or the stretching and wagging vibration of CH2, and to overtone or combination bands. Interestingly, we also show that the Raman spectrum of synthetic trichochrome F, a pigment that may be produced along with pheomelanin during pheomelanogenesis, is different from that of pheomelanin and similar to the spectrum of eumelanin. We could detect Raman signal of both eumelanin and pheomelanin in feathers and hairs where both pigments simultaneously occur without the need of isolating the pigment. This indicates that Raman spectroscopy represents a non-invasive method to detect pheomelanin and distinguish it from other pigments. This may be especially relevant to detect pheomelanin in animal skin including humans, where it has been associated with animal appearance and classification, human phototypes, prevention of skin diseases and cancer risk.

Retinal involvement of Paracoccioidomycosis: A Case Report.

PURPOSE: to describe the clinicopathologic features and treatment of a rare case of systemic paracoccidioidomycosis with choroidal and retinal involvement. DESIGN: retrospective interventional case report. PARTICIPANT: A 36-year-old young man with visual impairment in left eye with anterior uveitis and presence of whitish perimacular choroidal nodule, multiple underlying whitish spots and mid-periphery exudative retinal detachment. A primary extensive work-up for systemic infectious, autoimmune, neoplasic or inflammatory conditions was performed and high-resolution computer tomography scan demonstrated asymmetric parietal thickening of the trachea and bilateral diffuse multiple lobular opacities. Pulmonary bronchoscopy/biopsy of larynx, trachea and bronchial tube were also performed. Histopathological evaluation showed characteristic of Paracoccidioidomycosis. INTERVENTION: Patient was treated with oral sulphadiazine (1.5 g/day). MAIN OUTCOME MEASURES: Anterior uveitis, retinal examination, histopathological evaluation and primary clinical outcome were observed during systemic treatment. RESULTS: After 3 months of irregular treatment, choroidal lesions decreased in size forming atrophic scars and fibrotic spots; however visual acuity did not show any improvement. CONCLUSION: We report a rare case of systemic paracoccidioidomycosis with choroidal and retinal involvement treated with oral sulphadiazine.

New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins.

Tyrosinases are widely distributed in nature. They are copper-containing oxidases belonging to the type 3 copper protein family, together with catechol oxidases and haemocyanins. Tyrosinases are essential enzymes in melanin biosynthesis and therefore responsible for pigmentation of skin and hair in mammals, where two more enzymes, the tyrosinase-related proteins (Tyrps), participate in the pathway. The structure and catalytic mechanism of mammalian tyrosinases have been extensively studied but they are not completely understood because of the lack of information on the tertiary structure. The availability of crystallographic data of one plant catechol oxidase and one bacterial tyrosinase has improved the model of the three-dimensional structure of the active site of the enzyme. Furthermore, sequence comparison of tyrosinase and the Tyrps reveals that the three orthologue proteins share many key structural features, because of their common origin from an ancestral gene, although the specific residues responsible for their different catalytic capabilities have not been identified yet. This review summarizes our current knowledge of tyrosinase and Tyrps structure and function and describes the catalytic mechanism of tyrosinase and Dct/Tyrp2, which are better characterized.

[Penile leiomyosarcoma: case report and bibliographic review]. / Leiomiosarcoma de pene: presentación de un caso y revisión de la literatura.

OBJECTIVE: Primary penile leiomyosarcoma is a rare entity. Since 1930 only 30 cases have been reported in the bibliography. We wanted to add a new case to the international literature, in addition to a review of all available publications on the topic from 1957. A 54-year-old patient presented with a lobulated 8x4x3 cm lesion in the balanopreputial groove over two years; he was treated initially with partial penectomy and subsequently with total penectomy We analyze the evolution, progression and adjuvant treatments of this rare pathology. METHODS/RESULTS: We report one case with its clinical presentation, diagnostic tests performed for staging, treatment and follow-up. CONCLUSIONS: The first case of penile leiomyosarcoma was described by Levi in 1930. In 1957 Ashley and Edwards reported the first case in the British literature and in 1963 Pack reported the first in the American literature. MacKenzie et al. were the first to recognize two types of leiomyosarcoma: superficial and deep. The first develop from smooth muscle cells from the superficial dermal layers of the glans penis or distal third of the penis, they are generally asymptomatic and less malignant, rarely invading deeper structures and without involvement of the urethra on physical exam. The best prognostic predictors are type of tumor at presentation (superficial vs. deep) and treatment choice, being total penectomy more effective for failures of local resections than for primary treatment of deep tumors.

[Wilms tumor in an adult patient: case report]. / Tumor de Wilms en paciente adulto presentación de un caso.

Nephroblastoma or Wilms tumor is the most common renal neoplasia in children, representing 1/5 of the malignant tumors in this group. Nevertheless, the incidence of such tumor in adults is much rarer with less than 250 cases reported. Due to the low-frequency of this pathology in adults there is not a world widely accepted treatment modality. Currently, the therapeutic options derive from the National Wilms Tumor Study (NWTS). We report a new case with the radiological images, histologic findings, outcomes and follow-up.

A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio.

The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature 415, 497-502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil-borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.

Polyphenol oxidase activity expression in Ralstonia solanacearum.

Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to L-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.

Usefulness of four prognostic indices in differentiated thyroid cancer.

OBJECTIVE: To report the follow up of 82 patients with differentiated thyroid cancer in order to evaluate patients' clinical and tumor characteristics of prognostic value. PATIENTS AND METHODS: A prospective and comparative study was performed in 82 patients with differentiated thyroid cancer followed for a mean of 84 months. They were classified and included into four prognostic indices (pTNM, DeGroot's, AGES and NTCTCS) stages I and II were considered as a low risk ones and stages III and IV as a high risk group. At the end of the follow up period the patients were evaluated and divided into two groups, those with tumor activity (persistence or recurrence) and those in remission. A correlation coefficient and concordance analysis between the prognostic indices was done. Sensitivity, specificity and positive and negative predictive values were calculated in accordance with the tumor activity or the remission status. RESULTS: The highest correlation coefficient (r = 0.91, CI 0.85 to 0.94) was found between NTCTCS and pTNM indexes. The highest sensitivity (78%) and specificity (85.7%) was obtained with the NTCTCS index. CONCLUSION: In our population, NTCTCS index was a good option to evaluate the prognosis of patients with differentiated thyroid cancer.

Identification of an operon involved in tyrosinase activity and melanin synthesis in Marinomonas mediterranea.

The genomic region of Marinomonas mediterranea containing the genes required for tyrosinase activity and melanin synthesis has been cloned by marker rescue using the transposon-generated, amelanogenic strain T105. Five ORFs, two incomplete and three complete, have been sequenced in the genomic region where the transposon was inserted. RT-PCR analysis indicates that ORF 3, coding for tyrosinase, and ORF4, coding for a protein of 250 amino acids, are in the same transcriptional unit, constituting an operon whose promoter region has been determined by 5'-RACE. This operon has been sequenced in the wild-type and several mutant strains, indicating that both ORFs are required for expression of tyrosinase activity and melanin synthesis. The nitrosoguanidine generated, amelanogenic mutant ng56, shows a nonsense mutation in ORF3 coding for the tyrosinase. On the other hand, in the strain T105 the transposon is inserted in ORF4. The product of this gene is related to copper metabolism, since the addition of this metal ion to cell extracts or culture media partially restores melanin synthesis and tyrosinase activity in the strain T105. However, it does not show significant sequence similarity to previously characterized metallochaperones and hence may be an example of a new kind of those proteins. The operon has been denoted as ppoB, taking into consideration that ppoA denotes the M. mediterranea gene coding for the previously cloned polyphenol oxidase with laccase activity. This is the first demonstration of the tyrosinase gene forming part of an operon in a Gram-negative bacterium.

Melatonin ameliorates renal ischemia/reperfusion injury.

BACKGROUND: We studied whether melatonin is able to reduce organ damage during renal ischemia/reperfusion via its effects on the oxidative response in early and late reperfusion. MATERIALS AND METHODS: Renal ischemia/reperfusion injury (I/R) was induced in two groups of rats by 75 min occlusion of the left renal artery and vein and right nephrectomy, followed by reperfusion. The formation of reactive oxygen species was evaluated in the early reperfusion phase (60 min) by lipid peroxidation products and glutathione assay. In the late reperfusion phase (24 h) tissue neutrophil infiltration, inducible nitric oxide synthase (iNOS) gene expression, and histopathology were evaluated. Groups received either systemic melatonin (MEL) or normal saline (NS). There were two nonischemic sham control groups, one with and another without melatonin (S+MEL and S). RESULTS: Creatinine was higher in the NS group at all times. A reduction in glutathione and increases in lipid peroxidation products and myeloperoxidase activity induced by I/R indicated renal injury involving reactive oxygen formation. Melatonin reversed this oxidant response and reduced the rise in creatinine and iNOS expression. Seven-day group survivals were 5/10 for NS, 8/10 for MEL, and 10/10 for both Sham groups. CONCLUSIONS: Exogenous melatonin is able to preserve renal functional status following I/R-induced injury by increasing glutathione and reducing lipid peroxidation in the early reperfusion phase, without any apparent effect on neutrophil infiltration in the late reperfusion phase.

Synthesis and selective in vitro anti-melanoma effect of enantiomeric alpha-methyl- and alpha-ethyl-4-S-cysteaminylphenol.

Melanogenesis provides a unique target for the development of antitumour agents specific for malignant melanoma. Among the anti-melanoma compounds we have examined, 4-S-cysteaminylphenol (4-S-CAP), a phenolic amine, was found to have the most promising anti-melanoma effects. To further improve its efficacy as an anti-melanoma agent, we synthesized the R- and S-enantiomers (99% enantiomer excess) of alpha-methyl- 4-S-cysteaminylphenol (alpha-Me-4-S-CAP) and alpha-ethyl- 4-S-cysteaminylphenol (alpha-Et-4-S-CAP) by coupling 4-hydroxythiophenol with the oxazolines obtained from the (R)- and (S)-enantiomers of 2-amino-1-propanol and 2-amino-1-butanol, respectively. The enantiomers of alpha-Me-4-S-CAP and alpha-Et-4-S-CAP were found to be better substrates for tyrosinase than the natural substrate, L-tyrosine. In vitro experiments showed that all four enantiomers were highly cytotoxic to pigmented B16-F1 melanoma cells, the effect being 70-fold and 160-fold greater than that on non-pigmented B16-G4F melanoma cells and 3T3 fibroblasts, respectively. The cytotoxic effect against B16-F1 cells was completely inhibited by phenylthiourea, a tyrosinase inhibitor, or by N-acetyl-L-cysteine, which increases the intracellular reduced glutathione (GSH) level. 4-S-CAP and the enantiomers were taken up into B16-F1 cells at comparable rates, but showed varying rates of GSH depletion that were inversely correlated to the cytotoxicity. These results suggest that the use of enantiomers would increase the efficacy of tyrosinase-dependent cytotoxic phenols.

Marinomonas mediterranea is a lysogenic bacterium that synthesizes R-bodies.

The melanogenic marine bacterium Marinomonas mediterranea synthesizes R-bodies as revealed by transmission electron microscopy. These structures were previously described in some obligate symbionts of paramecia and some free-living bacteria, none of which was isolated from sea water. In other micro-organisms, the synthesis of R-bodies has been related to extrachromosomal elements. Accordingly, M. mediterranea induction by mitomycin C or UV radiation resulted in the production of defective phages resembling bacteriocins, indicating that it is a lysogenic bacterium. Two mitomycin-C-resistant strains defective in prophage replication have been isolated. These mutants, and the previously obtained strains ngC1, T102 and T103, the latter mutated in the ppoS gene encoding a sensor histidine kinase, are affected not only in phage replication but also in polyphenol oxidase activities and melanin synthesis, suggesting a relationship between the control of all these processes.

Conformation-dependent post-translational glycosylation of tyrosinase. Requirement of a specific interaction involving the CuB metal binding site.

Tyrosinase, the rate-limiting enzyme in mammalian melanogenesis, is a copper-containing transmembrane glycoprotein. Tyrosinase undergoes a complex post-translational processing before reaching the melanosomal membrane. This processing involves N-glycosylation in several sites, including one located in the CuB copper binding site, movement from the endoplasmic reticulum (ER) to the Golgi, copper binding, and sorting to the melanosome. Aberrant processing is causally related to the depigmented phenotype of human melanomas. Moreover, some forms of albinism and several other pigmentary syndromes are considered ER retention diseases or trafficking defects. A critical step in tyrosinase maturation is the acquisition of an ER export-competent conformation recognized positively by the ER quality control system. However, the minimal structural requirements allowing exit from the ER to the Golgi have not yet been identified for tyrosinase or other melanosomal proteins. We addressed this question by analyzing the enzymatic activity and glycosylation pattern of mouse tyrosinase point mutants and chimeric constructs, where selected portions of tyrosinase were replaced by the homologous fragments of the highly similar tyrosinase-related protein 1. We show that a completely inactive tyrosinase point mutant lacking a critical histidine residue involved in copper binding is nevertheless able to exit from the ER and undergo further processing. Moreover, we demonstrate that tyrosinase displays at least two sites whose glycosylation is post-translational and most likely conformation-dependent and that a highly specific interaction involving the CuB site is essential not only for correct glycosylation but also for exit from the ER and enzymatic activity.

Regulation of polyphenol oxidase activities and melanin synthesis in Marinomonas mediterranea: identification of ppoS, a gene encoding a sensor histidine kinase.

Marinomonas mediterranea is a melanogenic marine bacterium that expresses two different polyphenol oxidases. One of them is a multipotent laccase able to oxidize a wide range of substrates. The second enzyme is an SDS-activated tyrosinase. Using transposon mutagenesis, a mutant affected in the regulation of both polyphenol oxidase activities and melanogenesis has been isolated. The sequencing of the gene disrupted by the mini-Tn10 transposon in this mutant indicates that it encodes a hybrid sensor kinase. This sensor kinase shows three phosphorylated conserved domains: the transmitter domain containing a histidine site typical of sensor kinases, a receiver domain with an aspartate residue and an additional phosphotransferase domain with a second conserved histidine. This structural organization is characteristic of kinases participating in a phosphorelay system. Northern blot and lacZ operon fusions indicate that the multipotent laccase activity is regulated not only by PpoS but also by growth phase at the transcriptional level. These results suggest that PPO activities and melanin synthesis play a role in the adaptive response of M. mediterranea to stressful environmental conditions.

Molecular anatomy of tyrosinase and its related proteins: beyond the histidine-bound metal catalytic center.

The structure of tyrosinase (Tyr) is reviewed from a double point of view. On the one hand, by comparison of all Tyr found throughout nature, from prokaryotic organisms to mammals and on the other, by comparison with the tyrosinase related proteins (Tyrps) that appeared late in evolution, and are only found in higher animals. Their structures are reviewed as a whole rather than focused on the histidine (His)-bound metal active site, which is the part of the molecule common to all these proteins. The availability of crystallographic data of hemocyanins and recently of sweet potato catechol oxidase has improved the model of the three-dimensional structure of the Tyr family. Accordingly, Tyr has a higher structural disorder than hemocyanins, particularly at the CuA site. The active site seems to be characterized by the formation of a hydrophobic pocket with a number of conserved aromatic residues sited close to the well-known His. Other regions specific of the mammalian enzymes, such as the cytosolic C-terminal tail, the cysteine clusters, and the N-glycosylation sequons, are also discussed. The complete understanding of the Tyr copper-binding domain and the characterization of the residues determinant of the relative substrate affinities of the Tyrps will improve the design of targeted mutagenesis experiments to understand the different catalytic capabilities of Tyr and Tyrps. This may assist future aims, from the design of more efficient bacterial Tyr for biotechnological applications to the design of inhibitors of undesirable fruit browning in vegetables or of color skin modulators in animals.