[Why do patients come to emergency departments? Analysis of the use of the extra-hospital Emergency Service of Pamplona]. / ¿Por qué vienen los pacientes a urgencias? Análisis de la utilización del Servicio de Urgencias extrahospitalario de Pamplona.

OBJECTIVES: The demand in the extra-hospital emergency department of Pamplona has increased considerably in 2015 and 2016. The objective of the study is to determine the profile of the patients, the reasons why they come, Primary Care centres of origin, and if they have requested an appointment in them. MATERIAL AND METHODS: A multicentre, descriptive study using a self-completed questionnaire by patients was conducted during one week per month, between August 2016 and January 2017. The variables collected were: gender, age, time of evolution of the disease, Primary Care Centre of origin, appointment request in Primary Care Centre, time of delay until the appointment, and reason for going to the extra-hospital emergency department. The association between the call made to the Primary Care Centre and the rest of the variables was analysed using the Pearson χy test. RESULTS: A total of 3489 questionnaires were collected, with 61.10% of the respondents being women, and 76.1% were between 15 and 55 years old. Almost two-thirds (65.7%) had not requested an appointment in their Primary Care Centre. Those who had not called the Primary Care Centre (65.7%), referred to it being "sudden" (27.82%) and "due to work schedule problems" (19.21%). While the reasons for those who had called (33.21%) were "suggestion of the Primary Care Centre" (33.21%) and "have to wait for many days" (31.30%). CONCLUSIONS: Most patients, who come to the extra-hospital emergency department, do so without having previously requested an appointment in their Primary Care Centre, although this is the gateway to the health system. It is essential to educate the population about self-care and the way they should use health services.

The p53 tumor suppressor gene of the marsupial Monodelphis domestica: cloning of exons 4-11 and mutations in exons 5-8 in ultraviolet radiation-induced corneal sarcomas.

Inactivating p53 mutations are found in many ultraviolet radiation (UVR)-induced skin tumors. We examined 12 UVR-induced corneal tumors of the marsupial Monodelphis domestica for mutations in exons 5-8 of p53 and compared their mutational spectrum with that of UVR-induced skin tumors of other species. First we cloned and characterized a cDNA extending from the middle of exon 4 through exon 11 of the Monodelphis p53 gene. Based on the sequence information obtained, primers were designed to amplify introns 4-9 of the gene; intron primers to amplify individually exons 5-8 were subsequently developed. 'Cold' single strand conformational polymorphism analysis followed by reamplification of DNA with altered mobility and cycle sequencing revealed single p53 mutations in four of 12 tumors (33%), including one mutation in exon 5, two identical mutations in exon 7 and one mutation in exon 8. All mutations were at dipyrimidine sites and occurred on the non-transcribed strand. Three of the four were hallmark UVR-induced C-->T alterations. Three of the mutations were found at sites corresponding to human codons 248 and 273, which are mutational hotspots in human and murine UVR-induced squamous cell carcinomas. Our findings suggest that UVR-induced corneal sarcomas in Monodelphis will be valuable in studying mechanisms of p53 mutation in UVR-induced tumors.

Cloning and characterization of the CDKN2A and p19ARF genes from Monodelphis domestica.

The tumor suppressor gene, CDKN2A (p16), encodes a cyclin-dependent kinase inhibitor and functions as a negative regulator in the retinoblastoma pathway that blocks cell cycle progression from the G1 phase. The gene has been found to be deleted, truncated, mutated, or silenced by promoter methylation in a wide range of tumor types. Where melanoma CDKN2A mutations have been characterized, C --> T and CC --> TT transitions were found, indicating a direct role for ultraviolet radiation (UVR)-induced pyrimidine dimers in the formation of some tumors. The South American opossum, Monodelphis domestica, has been shown by our group and others to be susceptible to the induction of melanoma on chronic exposure to UVR alone. The CDKN2A gene and its exon 1beta alternate transcript p19ARF were cloned and sequenced from M. domestica to investigate the role of these genes in the development of UVR-induced melanoma and non-melanoma tumors. Both genes were first amplified by polymerase chain reaction (PCR) using cDNA from an opossum corneal-tumor cell-line library and degenerate primers based on human, mouse, and rat CDKN2A gene sequences. To verify these as normal sequences, both genes were then RT-PCR amplified from cultured normal opossum melanocyte mRNA. When comparing the tumor and melanocyte sequences, we found a UVR signature point mutation, a C --> T transition, within exon 2 in the corneal tumor cell line. The same mutation at this site in other tumors has been shown to alter the CDKN2A protein's ability to bind CDK4 kinase, which may lead to uncontrolled cell cycling. A comparison of the amino acid sequence of opossum CDKN2A showed identities relative to human, mouse, and rat between 57% and 63%, and when conserved amino acid substitutions are considered (similarity), the range is 63% to 67%. The amino acid identity and similarity for p19ARF ranged from 39% to 49%.

Oxidant inhibition of alphaLbeta2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage.

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.

H-ras oncogene activation in invasive UVR-induced corneal sarcomas of the opossum Monodelphis domestica.

Chronic exposure to ultraviolet radiation (UVR) induces corneal sarcomas in the South American opossum Monodelphis domestica. Cell lines are readily established from these tumors. Northern blotting of mRNA from six such cell lines revealed high expression of the H-ras oncogene. H-ras cDNA from an eye tumor cell line was cloned and characterized; the germline sequence of codons 12, 13, and 61 was confirmed by examination of H-ras sequences amplified from liver DNA by the polymerase chain reaction. The Monodelphis H-ras coding sequence is 84-89% identical to that of other vertebrates at the nucleotide level, and the predicted 189-amino-acid sequence differs by 2-12 amino acids from that of other vertebrates. Analysis of 12 primary invasive corneal sarcomas induced by chronic UVR exposure revealed no evidence of H-ras gene amplification or rearrangement. One tumor was heterozygous for an activating point mutation in codon 61 of the H-ras gene; the tumor was also homozygous for a point mutation at an adjacent site in codon 62. These results provide additional evidence for the functional importance and consequent evolutionary conservation of the ras oncogenes.

Photorepair of ultraviolet radiation (UVR)-induced pyrimidine dimers in lens epithelial DNA of Monodelphis domestica.

The repair of UV radiation-induced pyrimidine dimers has been measured in lens epithelial DNA of the marsupial Monodelphis domestica using a pyrimidine dimer-specific endonuclease from Micrococcus luteus. Approximately 40% of the initially induced dimers were repaired during 90 min exposures to photoreactivating light. This capacity of the lens epithelium to photorepair pyrimidine dimers may provide a means with which to determine whether pyrimidine dimers in lens epithelial DNA are involved in UV radiation-induced pathologic changes of the lens.

Localization and DNA sequence of a replication origin in the rhodopsin gene locus of Chinese hamster cells.

A chromosomal origin of DNA replication has been localized within the single-copy rhodopsin gene locus in Chinese hamster (line CHO) cells using two methods. In the first method, single-copy segments were identified at 3 to 15 kb intervals within approximately 75 kb (kb = 10(3) bases) of cloned genomic DNA containing the early-replicating rhodopsin gene near its middle. The cloned single-copy segments were then used as hybridization probes to quantify the replication of their corresponding genomic segments as synchronized cells progressed into S phase. In the second method, genomic DNA synthesized in vivo or in permeabilized early S phase cells was hybridized with slot-blots of the cloned single-copy DNA segments to identify the earliest replicating part of the 75 kb mapped region. The first method indicates that the earliest replicating DNA is located within a 10 kb region beginning 4 kb upstream from and extending 1 kb beyond the rhodopsin gene. The second method confirms the location in the vicinity of the rhodopsin gene and indicates that the earliest replicating region is located within or very near the 4.5 kb rhodopsin gene itself. An extended region of 12 kb that encompasses the entire early-replicating region has been sequenced for analysis and comparison with currently characterized origin regions associated with the CHO dihydrofolate reductase (dhfr) and human c-myc genes. There are several sequence similarities between the dhfr rhodopsin origin regions, including common transcription promoter consensus sequences, rodent Alu repeats with their 3'-A+T rich flanking sequences, A+T-rich yeast ARS and Drosophila SAR consensus sequences, and simple (GA)n repeats, but there are no extended regions of direct similarity. The rhodopsin gene locus is the second sequenced CHO origin region.

DNA contents of replication without DNA density labeling.

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.

UV induced (6-4) photoproducts are distributed differently than cyclobutane dimers in nucleosomes.

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.

Photofootprint of nucleosome core DNA in intact chromatin having different structural states.

Recently, we reported that the distribution of ultraviolet light (u.v.) induced pyrimidine dimers in nucleosome core DNA has a striking 10.3(+/- 0.1) base periodicity and the regions of enhanced quantum yield map to positions where DNA strands are farthest from the core histone surface. Improvement of the mapping procedure has allowed us to analyze this distribution in more detail, and compare the distribution pattern for nucleosome cores from intact chromatin having different higher-order structures (from the 10 nm filament to the 30 nm fiber). At all levels of chromatin compaction, we observed the following. (1) The average periodicity in pyrimidine dimer yield is 10.3 bases. (2) The peak-to-peak spacing in this distribution is significantly different from 10.3 bases in the region covering three helix turns immediately 5' of the dyad axis. (3) There is a suppression of photoproduct formation in the region of the dyad axis, especially at position 84 from the 5' end. (4) The approximately 10 base ensembles have alternating peak intensities throughout core DNA. Furthermore, peak deconvolution analysis of the pyrimidine dimer pattern yielded a striking similarity in photoproduct yield for the different levels of chromatin compaction. Irradiation of isolated core DNA yields a much more random distribution of photoproducts, although a weak modulation pattern is observed (indicating that there is a non-random alignment of adjacent pyrimidines in our core DNA preparations). This pattern includes a depression in photoproduct yield near position 95, suggesting that the sequence in this region plays a role in nucleosome positioning. These results show that the u.v. photofootprint is a sensitive, diagnostic probe of core histone-DNA interactions in intact chromatin, and these interactions are not significantly altered by changes in the structural state of the chromatin fiber.

UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro.

We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.

UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases.

We have determined the distribution of the major UV-induced photoproducts in nucleosome core DNA using the 3'----5' exonuclease activity of T4 DNA polymerase, which has been shown to stop digestion immediately 3' to UV-induced pyrimidine dimers. This assay is extremely sensitive since all DNA fragments without photoproducts (background) are reduced to small oligonucleotides, which can be separated from those fragments containing photoproducts. The results show that the distribution of UV-induced photoproducts (primarily cyclobutane dipyrimidines) is not uniform throughout core DNA but displays a striking 10.3 (+/- 0.1) base periodicity. Furthermore, this characteristic distribution of photoproducts was obtained regardless of whether nucleosome core DNA was isolated from UV-irradiated intact chromatin fibers, histone H1-depleted chromatin fibers, isolated mononucleosomes, or cells in culture. The yield of pyrimidine dimers along the DNA seems to be modulated in a manner that reflects structural features of the nucleosome unit, possibly core histone-DNA interactions, since this pattern was not obtained for UV-irradiated core DNA either free in solution or bound tightly to calcium phosphate crystals. Based on their location relative to DNase I cutting sites, the sites of maximum pyrimidine dimer formation in core DNA mapped to positions where the phosphate backbone is farthest from the core histone surface. These results indicate that within the core region of nucleosomes, histone-DNA interactions significantly alter the quantum yield of cyclobutane dipyrimidines, possibly by restraining conformational changes in the DNA helix required for formation of these photoproducts.

Comparison of the pharmacological profiles of cyclosporine, (Nva2)-cyclosporine and (Val2)dihydro-cyclosporine.

The pharmacological profiles of two new derivatives of the immunosuppressive drug, cyclosporine, is presented here. (Nva2)-CS has very similar properties to CS, but lacks the nephrotoxic side-effects. This derivative appears to be a potential successor to cyclosporine. (Val2)DH-CS seems to have a different spectrum of activities. It does not suppress humoral immunity and allograft rejection, but suppresses some types of cell-mediated immune responses. This derivative may prove useful in autoimmune situations where T cells are involved in the disease process.

Effect of pyrithiamine treatment on potassium ion fluxes in rat cortical slices.

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enhanced Na+/K+ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K+ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K+ was monitored directly with a K+-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K+ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K+ upon tetrodotoxin addition was inversely related to the extracellular Ca2+ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants.

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl(2) for maximum activity but was inhibited by MnCl(2); its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K(m) values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 mum and 11 mum; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5'-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5'-phosphate and 5-fluorouridine 5'-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.