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Objectives. This paper presents the development of a local sexual assault investigation kit (SAK) that doctors in the Philippines could use to collect biological samples from victims of sexual abuse, including child patients, that would be used for DNA testing. The study also reports on a management system via courier service to protect the integrity of the samples that could be eventually used as evidence in court from the collection site to the laboratory with sufficient backup measures. Methods. Women and Child Protection Units (WCPU) from Manila, Baguio, Cebu, and Davao partnered with the DNA Analysis Laboratory, Natural Sciences Research Institute of UP Diliman (NSRI-UPD) DNA Analysis Laboratory in testing the utility of a prototype SAK for the collection of biological samples from child patients. From January 2002 to March 2006, samples were collected from patients who went to WCPU within 72 hours post-contact and consented to participate in the study. WCPU doctors collected biological samples guided by the patient’s narratives and packaged the samples while following detailed documentation and chain of custody procedures. SAKs were then sent via a designated courier service from WCPU to the NSRI-UPD DNA Analysis Laboratory for DNA testing. The WCPU kept half of the samples collected, following recommendations made during sectoral consultations that included members of the Research Group of the Philippine Judicial Academy, prosecutors, and defense counsels. Case samples were packed well by the WCPU and received at the NSRI-UPD DNA Analysis Laboratory. Due to budget limitations, only the internal genitalia and patients’ reference buccal swabs were subjected to DNA tests as reported by Maiquilla et al.1 The remaining SAK components and case records were kept in a dedicated and secure storage facility. DNA testing reports were sent to the WCPU, which released them to the child patients and their legal guardians. Results. One hundred fifty-four female children aged 2-18 years old and their legal guardians agreed to participate in the study. Based on the initial interviews of the social workers who conducted the evaluation, all the participants came from families with very low socioeconomic status. The WCPU doctors then complied with prescribed procedures. To date, NSRI-UPD DNA Analysis Laboratory records show that a subpoena for expert testimony had been issued in only one case out of the 63 cases (1.6%) that were positive for male DNA. No further information was available on the final decision in this case due to the absence of any order from the judge granting the laboratory access to court records. Likewise, WCPUs did not have any information on the remaining 62 cases that could have used the DNA test results as evidence if a case had been filed in court. Conclusion. This study is the first to report the development and validation of a sexual assault investigation kit in the Philippines aimed at helping medical doctors in collecting and preserving critical biological samples for DNA testing. Using a dedicated courier service to send SAK from collecting agencies to the laboratory for DNA testing was successfully tested and resulted in faster delivery and significantly reduced overall cost. While DNA testing remains the most powerful tool for human identification and the technology has been available in the Philippines since 1997, certain factors have prevented it from being used routinely in sexual assault investigations, including those involving children.

This study aimed to evaluate the behavior and thermal comfort of 20 Girolando cows (5/8-H/G), with light and dark coats, in the wettest period of the year, in Santarém, Pará, Brazil, in pasture with access to shade, and plenty of drinking water and mineral salt. Animal behavior categories were computed for 12 h a day, on 3 days in a row, by trained observers. Three day shifts were considered: Morning (6:00 a.m. to 9:55 a.m.), Intermediate (10:00 a.m. to 01:55 p.m.) and Afternoon (2:00 p.m. to 05:55 p.m.). The Temperature Index (TI), the Black Globe Humidity Index (BGHI) and the Comfort Index (CI) were calculated to measure thermal comfort. At all times studied, BGHI pointed that the environment was outside the thermal comfort zone. Dark-coated animals spent more 34.26% of the time in activities in the shade. The light-coated animals remained more 11.88% of the time in the sun, performing their natural behaviors. Both light and dark coat animals remained more 77 and 74.44% of the time in the sun, respectively. The behavior “in the sun while grazing” was the most evident, in both coats, in the studied shifts. The behaviors “in the shade while walking” and “in the shade while standing idle” were more evident (p < 0.01) in dark-coated cattle. The grazing behavior was higher in animals with dark coat (p < 0.05). In all evaluated shifts, there was a positive correlation between the behavior “in the sun while grazing” with the CI (r = 0.44211; p < 0.0305). Behaviors performed in the shade, such as “idleness while lying down,” “ruminating while lying down and standing up,” and behaviors “in the sun,” “idleness while lying down” and “ruminating while lying down,” were negatively correlated with CI. It is concluded that, even in the wettest period of the year, in the Eastern Amazon, Girolando dairy cows are exposed to hot environments, which causes thermal discomfort and changes in their natural behavior, as they spend more time standing in shaded areas, usually in rumination. Also, light-coated cows spend more time in the sun, while dark-coated cows spend more time in the shade. Thus, light-coated cows tend to have health and zootechnical performance negatively affected.

The prosthesis installed immediately after extraction of natural teeth provides the patient with esthetics and function, avoids the loss of vertical dimension of occlusion and reestablishes the masticatory function, preventing future dysfunctions in the temporomandibular joint, making possible the reintegration of the patient in social life. In this context, the objective this study was report the manufacture of an immediate removable partial denture on the same day that the patient would undergo multiple dental extractions in aesthetic region, thus avoiding social and psychological disorders due to privation of teeth. The result considered extraordinary restoring the patient's well-being.

A sojicultura vem crescendo em cultivos agrícolas, principalmente nas áreas que estavam com pecuária extensiva em vias de degradação na região Amazônica. A reintegração dessas áreas ao sistema produtivo tem proporcionado ganhos econômicos, inclusive associados aos ganhos educacionais, devida a consolidação de universidades no oeste do Pará, após assinatura da moratória da soja, ampliando-se a oferta de emprego com a instalação de novas empresas na região. Os agricultores têm buscado alternativas a fim de atenderem as demandas relacionadas ao fornecimento de sementes, adubos e, sobretudo, quanto ao aporte tecnológico, para garantir o processo produtivo, em cada ano, da safra de grãos. O objetivo neste trabalho foi avaliar a produtividade de cultivares de soja associadas à graus-dias acumulados para subsidiar estratégias quanto a época de semeadura em cultivos de soja sob condições agrometeorológicas em Belterra (PA). Foram analisados dados de produtividades de diferentes cultivares com base em dados obtidos de experimento de campo em Belterra, onde foram fornecidas informações de dias dos estádios fenológicos (germinação, floração e colheita). Dados meteorológicos diários foram tratados, sendo esses oriundos da estação do Instituto Nacional de Meteorologia (INMET). Em planilha eletrônica (Excel), foram realizados os cálculos de graus-dia acumulados, adotando a temperatura base da soja de 10°C. O rendimento de grãos foi de 3,120kg.ha-1 na cultivar B, e o mais elevado foi de 3,240kg.ha-1, na cultivar C. Ao avaliar os valores acumulados de graus-dia até a floração, a cultivar C obteve a maior soma térmica (574°C) e a cultivar B contabilizou os menores valores de 515,85°C. A cultivar C, por ser de clico mais longo em relação as demais, contabilizou 1742°C, refletindo nas maiores produtividades. O total de graus-dia para completar o ciclo na cultivar C é indicativo de expressão em produtividade superior as demais cultivares analisadas, sendo uma variável resposta que deve ser considerada em planejamento de cultivos de soja para as condições de Belterra, importante município do polo de grãos do oeste do Pará. Made available in DSpace on 2019-09-19T00:36:48Z (GMT). No. of bitstreams: 1 Rica20191.pdf: 1130753 bytes, checksum: be68d07028596199cc865a8cfcb538e5 (MD5) Previous issue date: 2018

Treballs Finals de Grau d'Història de l'Art, Facultat de Geografia i Història, Universitat de Barcelona, Curs: 2020-2021, Tutor: Josep Lluís Falcó, [cat] El present treball se centra en els valors familiars hegemònics en la societat espanyola dels anys seixanta a través de la pel·lícula La gran familia (1962). El context espanyol de la dècada dels seixanta està protagonitzat per una sèrie de canvis com l’afany de modernitat i les reformes per l’obertura al món. Però, a la vegada, encara s’arrosseguen els valors tradicionals que havien acompanyat la societat espanyola dels quaranta i els cinquanta. El panorama cinematogràfic reflecteix aquest context canviant amb produccions diferents les unes de les altres. Mentre alguns directors ja miren cap al futur, d’altres segueixen vivint en el passat. L’estudi, doncs, tractarà el context social i polític del desarrollismo per entendre els valors familiars i la imatge de mare que apareix en l’exitosa pel·lícula de Fernando Palacios., [eng] The present work focuses on the hegemonic family values found in the spanish society of the sixties through the movie ‘La gran familia’ (1962). The spanish context of the sixties decade it’s led by a series of changes such as the eagerness of modernity and the political reforms for an opening to the world. But, at the same time, there are still being dragged traditional values that had accompanied the spanish society during the fourties and the fifties. The cinematographic scene reflects this changing context with varied productions. While some directors are looking to the future, others keep líving in the past. The study aims to acknowledge the social and political context of the ‘desarrollismo’ to understand the family values and the mother image that appear in the successful movie by Fernando Palacios.

Les effets therapeutiques des huiles essentielles (HE) sont largement dependants de leur composition chimique. Cette etude vise a mettre en evidence les molecules specifiques ou communes des HEs de resines et de les mettre en relation avec les effets therapeutiques indiques.

This study presents the first results obtained from a research that was carried out with university professors of an undergraduate degree program (undergraduate) in a public university of the State of Puebla, Mexico, with the purpose of investigating what concept they have about University Social Responsibility (USR) and what practices identify that have been carried out with students to promote it. In order to achieve this objective, a theoretical framework was created that would allow to have some references to identify which can be considered good practices in USR. Subsequently, semi-structured interviews were conducted with professors and several of them reported having promoted class projects linked to social needs; others talked about strategies implemented through the Mexican Social Service (mandatory), professional practices, volunteering, among others.

The analysis of different central nervous system regions is interesting and necessary as each one is involved in specific physiological processes and pathologies. On that matter, differences in the chemical composition between the same brain regions in different mice strains have been reported. In this sense, the development of a simple method for the identification of these regions depending on their chemical composition becomes relevant. Raman microspectroscopy, a non-destructive analytical chemical approach for biological samples, is a widely used method for qualitative, quantitative, and structural analysis in biochemical research. Ten brain structures in three different mice strains (Triple transgenic for Alzheimer Disease, 3xTgAD; Cluster 57 black 6, C57BL/6; and the Swiss strain, CD1) were analyzed, and variations among samples from several brain regions were found. Particularly, the pattern of signals from the hippocampus, the prefrontal and temporal cortices, the basal forebrain, the striatum, the cerebellum, and the hypothalamus was discernable. Interestingly, notable signals regarding non-peptide small neurotransmitters were observed, including those related to acetylcholine. These bands were present in the Raman spectra of the basal forebrain and prefrontal cortex in the three mice strains, consistent with the relative abundance of this neurotransmitter in those regions. However, signals with lower intensities appeared in the basal forebrain of C57BL/6 in comparison with the same tissue of the other two strains. In addition, the Raman intensity of bands assigned to catecholamines in the striatum was lower in the 3xTgAD than those from both CD1 and C57BL/6 mice strains. This approach, as well as the reported differences, has potential application for designing analysis on specific murine models of brain diseases.

A circuit controlling mechanical itch Considerable progress has been made in understanding and treating chemically induced itch. However, little is known about the mechanisms underlying mechanically evoked itch. Bourane et al. produced a model of mechanical itch by reducing the number of neuropeptide Y–expressing inhibitory spinal interneurons. This led to a selective increase in mechanically evoked itch-like behavior in mice. In contrast, chemically evoked itch or pain behavior remained unaffected. Science , this issue p. 550

Background: Lipid nanoparticles have been studied mainly as a means of transporting and releasing drugs. A special emphasis has been placed on designing nanoparticles that improve the delivery of drugs with targets in the central nervous system. Methods: The biomedical literature was searched for basic and clinical studies. The recent applications are described and related with their bioactivities. Results: The current review compiles data on the components and features of lipid nanoparticle systems as well as the necessary conditions for their selective action. As an example of their application, we present data from preclinical and clinical studies on lipid nanoparticles used as potent and efficient agents in the diagnosis and treatment of some neurodegenerative maladies, including Parkinson’s disease. Conclusion: Current evidence supports the application of lipid nanoparticles for designing carriers of drugs for neurodegenerative diseases. But also, we have gathered data that suggests a role of drug-free lipid nanoparticles as neuroprotective or preventive agents during neurodegenerative processes.

O objetivo neste estudo foi relatar as enfermidades que ocorrem em bezerros até um ano de idade, estabelecendo os principais fatores epidemiológicos associados à ocorrência dessas enfermidades. A fase de cria é considerada a mais sensível de todo o sistema produção. Os bezerros, principalmente nas primeiras semanas de vida são mais suscetíveis de adquirir doenças, sendo importante a atenção pelos tratadores quanto a cuidados básicos, dessa forma evitando as mais altas taxas de mortalidade que ocorrem principalmente até o desmane, sendo que os problemas mais frequentes são as infecções do umbigo, diarréias, pneumonias, verminoses e tristezas parasitárias. Diante do exposto, este estudo possibilitou por meio de análises de trabalhos científicos, concluir que os cuidados neonatais são extremamente necessários para evitar que o animal seja acometido por algum tipo de enfermidade, as condições do ambiente e o inadequado manejo sanitário associado á escassez de medidas profiláticas são determinantes para o aumento do índice de animais adoecidos. Por outro lado o manejo nutricional correto, prevenção e controle dessas doenças favorecem para melhorar a saúde, possibilitando que o animal expresse o seu máximo potencial para a produção e reprodução durante todo seu período de vida, contribuindo com um retorno econômico positivo, diminuindo os gastos com tratamentos e com futuras epidemias.

The innate immune cell network detects specific microbes and damages to cell integrity in order to coordinate and polarize the immune response against invading pathogens. In recent years, a cross-talk between microbial-sensing pathways and endoplasmic reticulum (ER) homeostasis has been discovered and have attracted the attention of many researchers from the inflammation field. Abnormal accumulation of proteins in the ER can be seen as a sign of cellular malfunction and triggers a collection of conserved emergency rescue pathways. These signalling cascades, which increase ER homeostasis and favour cell survival, are collectively known as the unfolded protein response (UPR). The induction or activation by microbial stimuli of several molecules linked to the ER stress response pathway have led to the conclusion that microbe sensing by immunocytes is generally associated with an UPR, which serves as a signal amplification cascade favouring inflammatory cytokines production. Induction of the UPR alone was shown to promote inflammation in different cellular and pathological models. Here we discuss how the innate immune and ER-signalling pathways intersect. Moreover, we propose that the induction of UPR-related molecules by microbial products does not necessarily reflect ER stress, but instead is an integral part of a specific transcription programme controlled by innate immunity receptors.

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3114–122, MAGE-C242–50, MAGE-C2336–344) or the standard proteasome (Melan-A26–35, tyrosinase369–377, gp100209–217). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2336–344 was only produced by intermediate proteasome β1i-β5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2191–200 is produced only by intermediate proteasome β1i-β5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

The laboratory evaluated 154 sexual assault cases from four Child Protection Units in the Philippines involving female child victims aged from 2 years to 18 years old. All child victims sought medical attention within 72 h after sexual contact. In 130 cases, the child victim knew the alleged offender and identified them during the interview with the social worker. Penile ejaculation was reported by 68 child victims with varying reports of washing after contact. Overall, 84 child victims admitted having wiped their genitalia prior to the collection of biological samples for DNA testing. Laboratory personnel examined vaginal smears in only 109 cases using a light microscope and reported 23 samples to be positive for sperm cells. Using the PowerPlex® short tandem repeat of the Y chromosome (Y-STR) DNA multiplex system, male DNA was detected in vaginal swab samples from 63 child victims. In 39 cases, positive amplification at 11 Y-STR DNA markers consistent with a single male DNA profile was observed. Twenty-eight of these full single Y-STR DNA profiles were found to be unique when searched in worldwide Y-STR DNA population databases (~40,000 haplotypes), eight haplotypes matching Filipinos and/or Asian haplotypes and one Y-STR DNA profile only matching European, Caucasian, and Latin American haplotypes. Y-STR DNA profiles generated will be compared with reference DNA profiles of alleged offenders once reference samples are submitted to the laboratory.

Peptide splicing is a newly described mode of production of antigenic peptides presented by MHC class I molecules, whereby two noncontiguous fragments of the parental protein are joined together after excision of the intervening segment. Three spliced peptides have been described. In two cases, splicing involved the excision of a short intervening segment of 4 or 6 aa and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of the other peptide fragment. For the third peptide, which is derived from fibroblast growth factor-5 (FGF-5), the splicing mechanism remains unknown. In this case, the intervening segment is 40 aa long. This much greater length made the transpeptidation model more difficult to envision. Therefore, we evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-aa-long precursor peptide. We evaluated the catalytic mechanism by incubating proteasomes with various precursor peptides. The results confirmed the transpeptidation model of splicing. By transfecting a series of mutant FGF-5 constructs, we observed that reducing the length of the intervening segment increased the production of the spliced peptide, as predicted by the transpeptidation model. Finally, we observed that trans-splicing (i.e., splicing of fragments from two distinct proteins) can occur in the cell, but with a much lower efficacy than splicing of fragments from the same protein.

Summary Animals depend on sensory feedback from mechanosensory afferents for the dynamic control of movement. This sensory feedback needs to be selectively modulated in a task- and context-dependent manner. Here, we show that inhibitory interneurons (INs) expressing the RORβ orphan nuclear receptor gate sensory feedback to the spinal motor system during walking and are required for the production of a fluid locomotor rhythm. Genetic manipulations that abrogate inhibitory RORβ IN function result in an ataxic gait characterized by exaggerated flexion movements and marked alterations to the step cycle. Inactivation of RORβ in inhibitory neurons leads to reduced presynaptic inhibition and changes to sensory-evoked reflexes, arguing that the RORβ inhibitory INs function to suppress the sensory transmission pathways that activate flexor motor reflexes and interfere with the ongoing locomotor program. Video Abstract

Cephalexin levels in the ejaculates of 31 infection-free volunteers have been determined following the administration of 1 g of antibiotic. In 16 subjects the cephalexin was administered by intramuscular injection and in 15 by mouth. In each case the volunteers were divided into groups of five each of which received the drug, 1, 2 or 4 hours before the collection of semen. Antibiotic concentrations were determined microbiologically on agar plates using Sarcina lutea ATCC 9341. For selection purposes healthy subjects were considered to be those showing a normal anamnesis and physical exploration, an absence of leucocytes demonstrable by a cytomorphological evaluation of the semen, semen citric acid and fructose levels within normal limits and a qualitatively and quantitatively similar saprophytic flora to that seen in a culture of urine taken immediately before the collection of semen. Maximum concentrations were obtained 2 hours after the administration of the antibiotic and the concentration following intramuscular injection (6.9 microgram/ml) was significantly greater than that following oral administration (4.7 microgram/ml).

CD8-positive T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. Here we describe a human minor histocompatibility antigen created by a polymorphism in theSP110nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order to that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20Sproteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation.

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EIItMan mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on σ54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.

Access to methylaluminoxane-type derivatives by a non-hydrolytic route is described. The procedure involves the reaction between trimethylaluminium (TMA) and benzoic acid (BA) at temperatures comprised from 20 to 100°C in toluene. The structure of the different aluminic derivatives formed for different ratios R=TMA/BA as well as the reaction pathways leading to these products were investigated by 1 H NMR spectroscopy at different temperatures. The monomeric and/or oligomeric Me 2 A1OA1Me 2 species issued from the reaction between BA and TMA were found to methylate and then activate (cationize) rac-At(Ind) 2 ZrCl 2 as shown by UV/visible spectroscopy analysis. This is further confirmed by successful hex-1-ene polymerisations carried out in the presence of such activators.

Leuconostoc mesenteroides Y105 and L. mesenteroides FR52 produce both mesentericin Y105 and B105, in equal amounts. The mesentericin operons of L. mesenteroides FR52 and Y105 which are involved in mesentericin Y105 and B105 production, were both sequenced and compared. Differences were limited to the two genes, mesD and mesE, which encode the dedicated transport system of mesentericin Y105. Analysis of mesentericin non-producing mutants and complementation experiments demonstrated that the major role of the membrane fusion protein, MesE, was in bacteriocin secretion for both strains. Moreover, the secretion machinery MesDE was demonstrated to be capable of transportation and maturation of the two pre-bacteriocins, mesentericin Y105 and B105. We also demonstrate that although MesDEs from strains Y105 and FR52 have significant sequence differences, both transporters were capable of assuring secretion of either bacteriocin.

The physiological significance of the presence of GLUT2 at the food-facing pole of intestinal cells is addressed by a study of fructose absorption in GLUT2-null and control mice submitted to different sugar diets. Confocal microscopy localization, protein and mRNA abundance, as well as tissue and membrane vesicle uptakes of fructose were assayed. GLUT2 was located in the basolateral membrane of mice fed a meal devoid of sugar or containing complex carbohydrates. In addition, the ingestion of a simple sugar meal promoted the massive recruitment of GLUT2 to the food-facing membrane. Fructose uptake in brush-border membrane vesicles from GLUT2-null mice was half that of wild-type mice and was similar to the cytochalasin B-insensitive component, i.e. GLUT5-mediated uptake. A 5 day consumption of sugar-rich diets increased fructose uptake fivefold in wild-type tissue rings when it only doubled in GLUT2-null tissue. GLUT5 was estimated to contribute to 100 % of total uptake in wild-type mice fed low-sugar diets, falling to 60 and 40 % with glucose and fructose diets respectively; the complement was ensured by GLUT2 activity. The results indicate that basal sugar uptake is mediated by the resident food-facing SGLT1 and GLUT5 transporters, whose mRNA abundances double in long-term dietary adaptation. We also observe that a large improvement of intestinal absorption is promoted by the transient recruitment of food-facing GLUT2, induced by the ingestion of a simple-sugar meal. Thus, GLUT2 and GLUT5 could exert complementary roles in adapting the absorption capacity of the intestine to occasional or repeated loads of dietary sugars.

We studied the role of the karyopherin a2 nuclear import carrier (also known as importin a2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin a2 accumu- lated massively in the cytoplasm within minutes of glu- cose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin a1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin a2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy- dependent. HepG2 and COS-7 cells displayed no glucose- induced GFP-karyopherin a2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyo- pherin a2 and the stimulation of glucose-induced gene transcription were simultaneously lost between pas- sages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin a2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

The σ 54 subunit of the RNA polymerase directs the expression of specific operons in association with cognate activators. Three different activators have been detected in the Listeria monocytogenes genome on the basis of the high conservation of a specific domain. Among them, the LacR activator, of the LevR family, was found just upstream from a newly described σ 54 –dependent operon, lpo , which presents a classical –24/–12 consensus promoter. The lpo operon encodes proteins similar to subunits of a PTS permease (EII) of the lactose family, namely LpoA (IIA) and LpoB (IIB). It also encodes a third putative protein, LpoO, with an unknown function but sharing high similarity with proteins also encoded within PTS operons from other bacteria and bearing a RGD motif. The expression of lpo was clearly dependent on LacR and σ 54 , and was induced by cellobiose, chitobiose and lactose. It underlies that the lpo operon likely encodes proteins involved in the utilization of these sugars by L. monocytogenes .

The strength improvement induced by addition of acetals such as methylal and ethylal in melamine–urea–formaldehyde (MUF) resins could be mostly ascribed to the increased effectiveness and participation of the melamine to resin cross-linking. This phenomenon has been shown here, by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, resin aging time stability, and mainly by laser light scattering, to be due to the following: (i) the increased solubility in water afforded by the acetals cosolvents of both the unreacted melamine and of the normally very much lower solubility, higher molecular weight, lower methylolated oligomers fraction, this leading to preferentially homogeneous and hence more effective reaction rather than heterogeneous reactions; and (ii) the effect that such acetals have on the size distribution of the resin colloidal particles, with the presence of acetals such as methylals markedly decreasing the average colloidal particles diameter of the resin. This latter effect appears to be due to the disruption of the molecular clustering of the MUF resin colloidal particles, but rearrangements in the size of the colloidal particles due to the decrease in surface tension of the system, which has also been noted, cannot be excluded. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1855–1862, 2002

SummarySensory circuits in the dorsal spinal cord integrate and transmit multiple cutaneous sensory modalities including the sense of light touch. Here, we identify a population of excitatory interneurons (INs) in the dorsal horn that are important for transmitting innocuous light touch sensation. These neurons express the ROR alpha (RORα) nuclear orphan receptor and are selectively innervated by cutaneous low threshold mechanoreceptors (LTMs). Targeted removal of RORα INs in the dorsal spinal cord leads to a marked reduction in behavioral responsiveness to light touch without affecting responses to noxious and itch stimuli. RORα IN-deficient mice also display a selective deficit in corrective foot movements. This phenotype, together with our demonstration that the RORα INs are innervated by corticospinal and vestibulospinal projection neurons, argues that the RORα INs direct corrective reflex movements by integrating touch information with descending motor commands from the cortex and cerebellum.

To analyze various approaches for the determination of surface free energies of solids from liquid-solid contact angles, comb-like polymers with controlled grafting rates and macromolecular structures have been synthesized. The surface free energy parameters were calculated from the contact angles of standard liquids on the solid surfaces. A mathematical approach of the so-called acid-base theory of adhesion was used to characterize the nucleophilic and/or electrophilic behavior of the polymeric solid surfaces. Thus, correlations were established between the macromolecular structures and the dispersive component of the surface free energy, on the one hand, and the acid and base components, on the other. The main conclusion is that the surface free energy components are relevant for the characterization of functional comb-like polymeric materials: the dispersive and base components increase with the number of grafted electron-donating groups, whereas the acid component decreases.

Cysteine proteinases of the papain superfamily, i.e., papain and cathepsin B-like proteinase, were found to be able to bind to laminin-coated wells. When papain and cathepsin B-like proteinase were used, saturable binding curves were found. The characterization of the binding site was carried out using synthetic peptides which corresponded to the most relevant functional sites of laminin and an octapeptide from the laminin binding region of the beta1 integrin subunit. In binding experiments, the decapeptide RNIAEIIKDI and the pentapeptide YIGSR were able to displace papain and cathepsin B-like proteinase from coated laminin. Nevertheless, the integrin beta1 peptide DLYYLMDL was the most powerful in the same experimental system. From these results, the C-terminal region of this cross-shaped protein, i.e., the end of the long arm, and the region including the YIGSR sequence of the short arm of the beta chain would be the cysteine proteinase binding site. This binding site is probably the result of the network organization of laminin which brings two regions, separated on a single laminin molecule, into proximity. In previous work, digestion of basement membranes has been found to be associated with the binding of cysteine proteinases to these supramolecular structures [N. Guinec, V. Dalet-Fumeron, and M. Pagano (1992) FEBS Lett. 308, 305-308]. The present report demonstrates that laminin is the cysteine proteinase binding protein of basement membranes. This property of laminin could be associated with tumor invasion and other tissue remodeling processes linked to proteolysis of basement membranes and extracellular matrices.

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 degrees C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-di nitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition-profile study showed a zinc-dependent collagenase activity. Using the 45-kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen-binding site, the same results were obtained. These results allow us to define a thiol-dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen-binding domain, a zinc-binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.

We describe the polymerase chain reaction (PCR) amplification of an 805 base pair fragment of 24S rRNA from the toxic dinoflagellate Dinophysis acuminata and the sequence of this fragment. We also describe a PCR-based assay for the specific detection of D. acuminata in seawater samples. Conserved primers, starting at positions 711 and 1489 of the 24S rRNA from the dinoflagellate Prorocentrum micans, were used for the PCR. The PCR product was cloned and sequenced. The fragment was aligned with rRNA sequences from other protists. Two oligonucleotides in variable domains of the sequence from D. acuminata were chosen and a protocol was defined for PCR-based detection of D. acuminata (30 cycles, 50∞C). Experiments conducted with seawater samples led to the detection of D. acuminata in naturally contaminated samples. The PCR enabled us to detect down to 30 cells/L seawater. The problem of interference from large concentrations of other phytoplankton species may be solved using nested PCR. Resume : Nous dOcrivons liamplification diun fragment de 805 paires de bases (pb) de liARN ribosomique 24S du dinoflagellO toxique Dinophysis acuminata. Nous dOcrivons Ogalement une mOthode de dOtection par rOaction de polymOrisation en chaOne (PCR) de Dinophysis acuminata dans lieau de mer. Les amorces conservOes au cours de liOvolution, dObutant en position 711 et 1489 sur liARN ribosomique 24S du dinoflagellO Prorocentrum micans, ont OtO utilisOes pour la PCR. Le produit de la rOaction a OtO clonO et sOquencO. Un fragment de 805 pb a OtO obtenu puis alignO avec les sOquences diARN ribosomique 24S diautres espces de protistes. Deux oligonuclOotides ont OtO choisis dans les domaines variables de la sOquence et un protocole a OtO mis au point afin de dOtecter liespce D. acuminata (30 cycles, 50∞C). Les expOrimentations rOalisOes avec lieau de mer nous ont permis de dOtecter D. acuminata dans des Ochantillons naturellement contaminOs. La PCR nous permet de dOtecter des concentrations aussi faibles que 30 cellules/L dieau de mer. Les interfOrences avec la prOsence diun nombre important diautres espces du phytoplancton peuvent Œtre OvitOes par liutilisation diune PCR interne.

Two hundred and thirteen Staphylococcus aureus and 51 other staphylococcal strains were isolated from 121 foodstuffs of current consumption and two cutaneous samples. Their ability to produce staphylococcal enterotoxins was tested and S. aureus strains were biotyped. The S. aureus strains (30.5%) produced at least one of the five known staphylococcal enterotoxins whereas coagulase negative staphylococci did not produce any of them. The raw milk cheeses analysed were primarily contaminated by strains belonging to animal or unspecified biovars. Only 15.9% of the S. aureus strains isolated from these products produced enterotoxins whereas 43% were found to be enterotoxigenic amongst the S. aureus strains isolated from the other foodstuffs. The scheme of biotyping used seems to be reliable, allowing the classification of 73.7% of the strains. S. aureus strains of human biovar origin were most often enterotoxigenic and enterotoxin C was the predominant type identified. It was produced by 66% of the enterotoxigenic strains, singly or in combination with other enterotoxins. Approximately 77% of the human strains also produced enterotoxin C, which is an amazing epidemiological distinctive feature of the strains studied. Moreover ELISA tests used in this work exhibit problems of specificity.

In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifically, new possibilities are explored in relation to allosteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson's disease, and on muscarinic receptors for Alzheimer's disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopamine receptor holds promise as a relevant therapeutic strategy for Parkinson's disease. Regarding the treatment of Alzheimer's disease, the design of dualsteric ligands for mono-oligomeric rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

We prove that the Lipschitz-free space over a countable compact metric space is isometric to a dual space and has the metric approximation property.

The efficacy and safety of rufloxacin (400 mg, single dose) were compared to those of norfloxacin (400 mg twice a day for 3 days) for the treatment of women with uncomplicated cystitis. In addition, urine levels, drug level/MIC ratio, and urine antibacterial activity 72 to 84 h after treatment initiation were determined in a subgroup of patients for pharmacodynamic assessment. A total of 203 women were included and treated in this open, randomized clinical trial; 100 patients received norfloxacin, whereas 103 received rufloxacin. Of these, 156 (74 and 82 patients in the norfloxacin and rufloxacin groups, respectively) were considered bacteriologically evaluable. At the first follow-up visits (3 to 12 days after starting the treatment), bacteriological cure rates were 99 and 94% for norfloxacin and rufloxacin, respectively. Seventy-nine percent (119 of 150) of bacteriologically cured patients attended a long-term follow-up visit (4 to 6 weeks after starting the treatment), where a relapse rate of 4% (2 of 54) and 5% (3 of 64) were found in the norfloxacin and rufloxacin groups, respectively. The pharmacodynamic evaluation performed in 35 patients showed similar median urine levels (approximately equal to 25 micrograms/ml) and urine antibacterial activity for both treatment groups against initial isolates, despite a higher norfloxacin level/MIC ratio due to the lower MIC of norfloxacin. Twenty-one patients (20%) in the rufloxacin group and 12 patients (12%) in the norfloxacin group reported 39 and 16 adverse events, respectively, almost all of them being mild and lasting < 24 h. Overall, gastrointestinal reactions were the most frequent adverse events reported. However, 12 patients treated with rufloxacin reported 15 central nervous system adverse events. This study shows that single doses of rufloxacin are as effective as a norfloxacin 3-day standard treatment in uncomplicated cystitis. The results obtained with rufloxacin are consistent with its pharmacodynamic properties.

A new commercial agar (Uricult-Trio) with 8-hydroxyquinoline-beta-glucuronide was used to assess 2,536 uropathogens for beta-glucuronidase activity typical of Escherichia coli. Included in the study were 1,807 strains of the family Enterobacteriaceae, 284 strains of nonfermentative bacilli, 345 strains of gram-positive cocci, and 100 yeast strains. In identifying E. coli, the test agar gave a sensitivity of 95.5% and a specificity of 97.2%. Fifty E. coli isolates gave negative reactions; 31 non-E. coli strains produced black colonies characteristic of E. coli. No growth of gram-positive cocci and no false-positive reactions from yeasts were observed. The recovery rate for E. coli on this agar was at least 10% higher than that on blood agar.

International audience; In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.

A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide- N -glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).

Peptide splicing allows the production of antigenic peptides composed of two fragments initially non-contiguous in the parental protein. The proposed mechanism of splicing is a transpeptidation occurring within the proteasome. Three spliced peptides, derived from FGF-5, melanoma protein gp100 and nuclear protein SP110, have been described. Here, we compared the production of these spliced peptides by the standard proteasome and the immunoproteasome. Differential isotope labelling was used to quantify (by mass spectrometry) the fragments contained in digests obtained with precursor peptides and purified proteasomes. The results show that both the standard and the immunoproteasomes can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are more efficiently produced by the standard proteasome, whereas the SP110 peptide is more efficiently produced by the immunoproteasome. This seems to result from differences in the production of the two splicing partners, which depends on a balance between cleavages liberating or destroying those fragments. By showing that splicing depends on the efficiency of production of the splicing partners, these results also support the transpeptidation model of peptide splicing. Furthermore, given the presence of immunoproteasomes in dendritic cells and cells exposed to IFN-γ, the findings may be relevant for vaccine design.

Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital-induced baboons (Papio papio). The cytochrome FA possesses the same N-terminal amino acid sequence as P450 MK2 from crab-eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 6 beta position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N-terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7-hydroxylase. Antibodies against P450 FA and FI cross-react with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five-times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51-kDa protein and a moderate inducer of the 49-kDa protein, while phenobarbital has the opposite effect on the two proteins.

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after a 48-h enrichment period. Using this technique, as little as 102 bacteria per ml of enrichment broth can be detected.

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450tu) crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49-kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well (R = 0.78) with the microsomal coumarin-7-hydroylase (COH) activity. This activity is strongly and completely inhibited by anti-P450tu antibody (IC50 = 0.13 mg IgG/mg microsomal protein). The crossreacting 49-kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7-ethoxycoumarin O-deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and beta-naphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a combination of aminooctyl-amino-Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N-terminal amino-acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.