Multiparametric magnetic resonance imaging and prostate cancer: what's new? / Resonancia magnética multiparamétrica y cáncer de próstata: ¿qué hay de nuevo?

Prostatic multi-parametric magnetic resonance imaging (MP-MRI) has recently had a wide development becoming a key tool in the diagnostic and therapeutic decisions in prostate cancer (Pca). The fast development both in technology and in reading (PIRADS V2) requires a continuous updating of knowledge within this area. The aim of this article is to present an updated revision of technical aspects, reading patterns and prostatic MP-MRI in Pca, with a multidisciplinary approach. Currently guidelines establish the use of the MP-MRI when there is a high PSA and a negative prostatic biopsy; tumor staging; evaluation in candidates to active surveillance; focal treatments plans and tumoral recurrence evaluation. Although it is used in other indications in some centers, like its use in patients suspicious of Pca but with no previous biopsy, there is still the need of a cost/benefit assessment for its use to be wider.

Selection of the method to appraise and compare health systems using risk stratification: the ASSEHS approach.

To face the challenge of active and healthy ageing, European Health Systems and services should move towards proactive, anticipatory and integrated care. The comparison of methods to combine results across studies and to determine an overall effect was undertaken by the EU project ASSEHS (Activation of Stratification Strategies and Results of the interventions on frail patients of Healthcare Services, EU project (No. 2013 12 04). The questions raised in ASSEHS are broad and involve a complex body of literature. Thus, systematic reviews are not appropriate. The most appropriate method appears to be scoping studies. In this paper, an updated method of scoping studies has been used to determine the questions needed to appraise the health systems and services for frailty in the ageing population. Three objectives were set (i) to detect a relevant number of risk stratification tools for frailty and identify the best-in-class, (ii) to understand the feasibility of introducing stratification tools and identify the difficulties of the process and (iii) to find evidence on the impact of risk stratification in Health Services. This novel approach may provide greater clarity about scoping study methodology and help enhance the methodological rigor with which authors undertake and report scoping studies.

WT1 mutations may be a cause of severe renal failure due to nephroblastomatosis in Wilms' tumor patients.

Wilms' tumor suppressor gene (WT1) encodes a transcription factor required for normal development of the genitourinary system. Germline WT1 mutations have been described in a wide spectrum of pathological conditions, including kidney diseases, genital abnormalities and Wilms' tumor. Here we report a 4-year-old male patient who presented with bilateral cryptorchidism, Wilms' tumor, nephroblastomatosis and renal failure without nephrotic proteinuria. Sequence analysis of the WT1 gene demonstrated a constitutional heterozygous nonsense mutation in exon 7, which leads to a truncation of the WT1 protein at the zinc-finger 1. In the DNA of the tumor, we observed the same mutation in homo/hemizygosity. Given the requirement of WT1 for normal development, the WT1 mutation is likely to be responsible for the nephroblastomatosis and, in consequence, for the severe renal failure observed in our patient. This finding extends the spectrum of kidney diseases related to WT1 mutations and points to the need to screen for this gene in children with genitourinary abnormalities and Wilms' tumor because of the associated risk of nephroblastomatosis and renal failure in those carrying WT1 mutations.

Human autoantibody to defensin: disease association with hyperreactive onchocerciasis (sowda).

Chronic hyperreactive onchodermatitis (sowda) is a severe form of onchocerciasis observed in a subset of individuals infected with the filarial nematode Onchocerca volvulus. SDS-PAGE and immunoblot analyses of O. volvulus adult worm extracts were used to characterize the antigens of the marked antibody response of sowda patients. One 2.5-kD antigen was recognized by sera from all 35(100%) sowda patients that were studied. In comparison, only 7 of 44 (16%) patients with generalized onchocerciasis and 11 of 21 (52%) of exposed individuals with no microfilariae in skin snips and no signs of disease showed reactivity to this antigen. Microfilaricidal treatment of sowda patients with improvement of the clinical status was associated with a decrease or disappearance of antibodies to the 2.5-kD antigen. Amino acid sequencing of the antigen indicated identity to human defensins 1-3 of neutrophils. Defensin was demonstrated by immunohistochemical staining in onchocercal nodules on the surface of adult filariae and in the surrounding tissue. A similar staining pattern was observed for other proteins present in neutrophils such as myeloperoxidase, elastase, and the L-1 protein complex (MRP 8/MRP 14), indicating that neutrophils, macrophages, and their proteins predominate in the environment adjacent to the worms. These results demonstrate an association between the presence of autoantibodies to defensins and an infectious disease of known etiology. The association with a particular form of onchocerciasis, sowda, suggests a link between formation of autoantibodies to defensin and enhanced immune reactivity towards the parasite.

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants.

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea.

Hydroxyurea reduces DNA replication by nucleotide deprivation, whereas UV damage generates DNA photoproducts that directly block replication fork progression. We show that the low fidelity class Y polymerase Pol eta is recruited to proliferating cell nuclear antigen at replication forks both by hydroxyurea and UV light. Under nucleotide deprivation, Pol eta allows cells to accumulate at the G1/S boundary by facilitating slow S-phase progression and promotes apoptosis. Normal cells consequently enter apoptosis at a faster rate than Pol eta-deficient cells. Coincident with hydroxyurea-induced S-phase delay, Pol eta-deficient cells undergo more replication fork breakage and accumulate more foci of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX. We conclude that under conditions of nucleotide deprivation, Pol eta is required for S-phase progression but is proapoptotic. However, as Pol eta is reported to require higher nucleotide concentrations than class B replicative polymerases, its recruitment by hydroxyurea requires it to function under suboptimal conditions. Our results suggest that hydroxyurea-induced apoptosis occurs at the G1/S boundary and that initiation of the S-phase requires greater nucleotide concentrations than does S-phase progression.

Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair.

Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth.

[Urothelial carcinoma in a woman with autosomal dominant polycystic kidney disease: description of a case and review of the literature]. / Carcinoma urotelial renal en una paciente con enfermedad renal poliquística autosómico dominante: presentación de un caso y revisión de la literatura.

We describe a case of urothelial carcinoma of renal pelvis in a 48 years old woman affected of autosomal dominant polycystic kidney disease (ADPKD). We discuss the difficulty of the radiological diagnostic and we revise the incidence of renal tumors in this entity. Association between urothelial carcinoma and ADPKD is highly infrequent and without apparently causal relation.

The structure of the carbohydrate units of the carboxyl-terminal peptide of procollagen as elucidated by 500 MHz 1H-NMR spectroscopy.

The oligosaccharides of chick embryo type I procollagen were isolated from carboxyl-terminal propeptide fragment by exhaustive digestion with papain and pronase, and then purified as a mixture of glycopeptides. The structures of the oligosaccharides were established by high-resolution 1H-NMR spectroscopy and found to be a mixture with respect to the non-reducing terminal residues as shown below: (formula; see text) The percentages refer to the relative amount of those mannose residues present in the mixture. The data suggest that the oligosaccharides are a microheterogeneous mixture of high-mannose type glycans containing between six and nine mannose residues per carbohydrate unit. Such carbohydrate chains, although not uncommon for glycoproteins, had never been found before for collagen or collagen-related compounds.

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin.

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.

[Intraocular lens (IOL) power calculation after keratorefractive procedures]. / Cálculo del poder dióptrico de la lente intraocular (LIO) tras cirugía refractiva.

PURPOSE: To compare various methods of estimating corneal power for IOL calculation for cataract surgery after corneal refractive surgery. METHODS: Review of the medical literature and case reports. RESULTS: For more accurate IOL power calculations we need pre- (Kpre) and post-treatment keratometry records (Kpost) if using 3rd generation formulae, or Kpost records when using 4th generation formulae. CONCLUSIONS: Prior to performing a keratorefractive procedure it is advisable to have pre-treatment refraction and keratometry (K and method) registered. 3rd generation formulae with Aramberri's double-K correction, or 4th generation formulae are recommended.

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer.

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.

Microanalysis of the amino acid sequence of the eclosion hormone from the tobacco hornworm Manduca sexta.

The amino acid sequence of the eclosion hormone from the tobacco hornworm Manduca sexta has been determined, using less than 500 pmol of protein and microanalytical techniques. The protein contains 62 amino acid residues and has a molecular mass of 6813 Da. The amino-terminal sequence is similar to that of a 13-residue segment at the amino terminus of the eclosion hormone of the silkworm Bombyx mori, but the hormone is not otherwise homologous with other hormones or proteins.

Time-resolved surface charge change on the cytoplasmic side of bacteriorhodopsin.

The pH-sensitive dye 5-iodoacetamidofluorescein was covalently bound to a single cysteine residue introduced by site-directed mutagenesis in position 101 on the cytoplasmic surface or in position 130 on the extracellular surface of the proton pump bacteriorhodopsin. Using time-resolved absorption spectroscopy at 495 nm a transient increase was observed in the apparent pK of the dye attached at residue 101. At pH 7.3 the rise and decay times of this pK-change (approximately 2 ms and approximately 60 ms) correlate well with decay times observed for the M and O intermediates and with the proton uptake time. Interpreting the pK-increase of +0.18 pH-unit in terms of a transiently more negative surface charge density, we calculate a change of -0.80 elementary charge per bacteriorhodopsin at the cytoplasmic surface. It is likely that this charge change is due to the transient deprotonation of aspartate-96. With the label in position 130 on the extracellular surface no transient pK-shift was detected.

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin.

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

Evidence for the repetitive domain structure of pig calpastatin as demonstrated by cloning of complementary DNA.

A clone of complementary DNA (cDNA) coding for pig heart calpastatin was isolated using synthetic oligonucleotide probes. The amino acid sequence deduced from the nucleotide sequence revealed the occurrence of a repetitive sequence at the interval of 140 amino acids, substantiating the multidomain structure of calpastatin. A portion of the sequence of 251 amino acid residues predicted for pig heart calpastatin (107 kDa) was found to be identical with that of a peptide fragment derived from pig erythrocyte calpastatin (68 kDa) and sequenced by Edman degradation.

Characterization and cloning of the gene encoding the vacuolar membrane protein EXP-2 from Plasmodium falciparum.

As a contribution to the characterization of the parasitophorous vacuolar membrane from Plasmodium falciparum we have begun the identification of vacuolar membrane proteins. Exported protein-2 (EXP-2) is a vacuolar membrane protein exposed into the vacuolar space. To further characterize EXP-2, it was purified, and the 45 N-terminal amino acids were determined by micro-sequencing. Based on this information, partial cDNA and genomic fragments were amplified by PCR and used as probes for the isolation of complete cDNA and genomic DNA clones. The single copy gene is located on chromosome 14, and is transcribed during the ring stage of parasite development. The open reading frame encodes an N-terminal signal sequence which is cleaved from the mature protein. The amino acid composition of EXP-2 is characterized by charged amino acids, with a high abundance of aspartate residues in the C-terminal portion of the protein. In contrast to EXP-1, an integral protein of the vacuolar membrane, EXP-2 lacks a typical hydrophobic transmembrane domain. We suggest that EXP-2 may associate with the vacuolar membrane via an amphipathic helix located in the N-terminal half of the protein.

Photoaffinity labeling of the NMDA receptor.

The structure of NMDA receptors in situ has been probed with the novel photoaffinity ligand 125I-CGP 55802A. By covalently linking the radioactive high-affinity photolabel to NMDA receptors in bovine brain we have identified a protein of 175 kDa associated with the binding site for NMDA receptor agonists and competitive antagonists. Based on its molecular size the photolabeled protein is likely to correspond to the NR2A and/or NR2B subunit. The photoaffinity ligand will permit the assessment of regulatory changes in NMDA receptor subunit expression.

Effects of mutagenetic substitution of prolines on the rate of deprotonation and reprotonation of the Schiff base during the photocycle of bacteriorhodopsin.

Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions of Pro-50, Pro-91 and Pro-186 on the deprotonation and reprotonation kinetics of the Schiff base (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate that Pro-50 and Pro-91, as well as Pro-186, are important for the proton-pumping function of bR.

Elucidation of intestinal transport results as a function of age.

Duodenal, jejunal, ileal and caecal morphometrics were determined in chickens from hatching to the age of 15 weeks. The ratios of fresh weight/surface area and dry weight/surface area showed age-dependent changes in all the intestinal segments studied. The percentage of mucosa in each segment was also age-dependent in the first three weeks of life with a higher participation in the proximal intestine. Transport of 3-oxy-methyl-D-glucose was determined in the jejunum and ileum using a perfusion technique in vivo and expressed as a function of fresh weight, dry weight and surface area. The age-dependent differences observed in weight and mucosa composition of the intestine make it difficult to interpret the results of transport capacity during development. The optimum expression of results in the field of intestinal absorption may be the surface area, since it involves the smallest errors.