Extracellular vesicles from neural progenitor cells promote functional recovery after stroke in mice with pharmacological inhibition of neurogenesis.

Neural progenitor cells (NPCs) of the subventricular zone proliferate in response to ischemic stroke in the adult mouse brain. Newly generated cells have been considered to influence recovery following a stroke. However, the mechanism underlying such protection is a matter of active study since it has been thought that proliferating NPCs mediate their protective effects by secreting soluble factors that promote recovery rather than neuronal replacement in the ischemic penumbra. We tested the hypothesis that this mechanism is mediated by the secretion of multimolecular complexes in extracellular vesicles (EVs). We found that the molecular influence of oxygen and glucose-deprived (OGD) NPCs-derived EVs is very limited in improving overt neurological alterations caused by stroke compared to our recently reported astrocyte-derived EVs. However, when we inhibited the ischemia-triggered proliferation of NPCs with the chronic administration of the DNA synthesis inhibitor Ara-C, the effect of NPC-derived EVs became evident, suggesting that the endogenous protection exerted by the proliferation of NPC is mainly carried out through a mechanism that involves the intercellular communication mediated by EVs. We analyzed the proteomic content of NPC-derived EVs cargo with label-free relative abundance mass spectrometry and identified several molecular mediators of neuronal recovery within these vesicles. Our findings indicate that NPC-derived EVs are protective against the ischemic cascade activated by stroke and, thus, hold significant therapeutic potential.

Preparation and ex vivo investigation of an injectable microparticulate formulation for gastrointestinal mucosa polyp resection.

Endoscopic submucosal dissection (ESD) and endoscopic submucosal resection (EMR) are non-invasive endoscopic techniques. They allow an early excised gastrointestinal (GI) mucosal precancerous lessions. For their application is necessary to use a submucosal injection that lifts the area to excise. The main objective of this study was the preparation of a microparticulate-based fluid for injection in the GI submucosa. Alginate microparticles (MPs) were developed by the solvent displacement technique and characterized by particle size, surface electrical properties, swelling, degradation, rheology, adhesion, leakage, syringeablity and stability. Furthermore, their potential to form a submucosal cushion was assayed in porcine stomach mucosa and porcine colon mucosa. Results showed MPs sizes below 160 µm, negative surface charge around -50 mV at pH = 6, high rates of swelling and good adhesion. The microparticulate-based fluid exhibited pseudoplastic behavior following the Ostwald-de Waele rheological model. A brief force is sufficient for its injection through a syringe. Finally, formulations were able to provide a submucosa elevation of 1.70 cm for more than 90 min and 120 min in the porcine stomach and colon, respectively.

Towards a collaborative structure of interpreter-mediated medical consultations: Complementing functions between healthcare interpreters and providers.

In today's multilingual and multicultural societies, healthcare interpreters are increasingly needed to mitigate communication barriers in language-discordant, intercultural medical consultations. To orient these interactions, existing guidelines, best practices and recommendations shed light on the behaviour and responsibilities of interpreters and healthcare providers involved. These documents, however, mainly treat both professionals as individuals that take care of separate, unrelated dimensions of consultations, thus failing to address how they can work collaboratively. This seems to be particularly relevant if we consider that prescriptive documents advocate for an invisible interpreter rather than an active participant, consequently ignoring the positive functions interpreters are playing when they step out of their prescribed roles. In this context, this paper sets out to explore potential collaboration between both professional groups to improve communication as a whole. Drawing on Goffman's production format (1981), we examined excerpts from real interpreter-mediated medical consultations that took place at a public hospital in Madrid (Spain) over a period of five months (February-June 2017). Data analysis reveals that interpreters enact an author role as main participants of consultations and serve several functions in medical encounters, consequently sharing some of the responsibilities which are conventionally seen as doctors'. This may reveal potential areas of interest for interprofessional collaboration. In addition to interpreting, participants performed other clinical functions, thus accounting for complementary functions of that performed by healthcare providers. Interpreters act as clinical and therapeutic allies, patient empowerers and metalinguistic negotiators. In light of our findings, the next step is to design a new model for the interpreter-mediated medical consultations that integrates both perspectives in a collaborative, non-excluding proposal.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy.

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

Efficacy and Safety of a Novel Submucosal Injection Solution for Resection of Gastrointestinal Lesions.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are minimally invasive and efficient techniques for the removal of gastrointestinal (GI) mucosal polyps. In both techniques, submucosal injection solutions are necessary for complete effectiveness and safety during the intervention to be obtained. The main objective of this study was to evaluate the efficacy and safety of a new sterile submucosal injection solution for EMR/ESD used within a clinical protocol in patients with intestinal polyps. We carried out a prospective study between 2016 and 2017 with patients who attended the Endoscopy Consultation-Digestive Department of Primary Hospital. Patients were selected for EMR/ESD after the application of clinical protocols. Thirty-six patients were selected (≥ 66 years with comorbidities and risk factors). Lesions were located mainly in the colon. Our solution presented an intestinal lift ≥ 60 min in EMR/ESD and a high expansion of tissue, optimum viscosity, and subsequent complete resorption. The genes S100A9 and TP53 presented an expression increase in the distal regions. TP53 and PCNA were the only genes whose expression was increased in polyp specimens vs. the surrounding tissue at the mRNA level. In EMR/ESD, our solution presented a prolonged effect at the intestinal level during all times of the intervention. Thus, our solution seems be an effective and safe alternative in cases of flat lesions in both techniques.

Novel Polymeric Formulation for Removal of Gastrointestinal Polyps by Digestive Endoscopy.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are two techniques used in the resection of gastrointestinal mucosal polyps. The aim of this work is the development and evaluation of an innovative polymeric solution containing sodium carboxymethylcellulose and hyaluronic acid. For this purpose, several mixtures of these two main components, as well as other components such as fructose, citric acid, and zinc, are evaluated in terms of physicochemical and microbiological properties, rheological behavior, extensibility, syringeability, and stability at different storage conditions. Furthermore, the potential production of mucosal elevation and duration is also studied by an ex vivo model using porcine stomach and colon. Results show that the developed polymeric solutions possess optimal values of pH, from 4.58 to 6.63, for their use in the gastrointestinal tract. The formulations exhibit both Newtonian and pseudoplastic behaviors with different viscosity values as a function of their composition. All formulations exhibit high stability properties and no bacterial or fungal growth is detected. MCS01 and MCS05 are the polymeric solutions with the best syringeability results. In this line, MCS05 is the formulation that provides the highest, 2.20 ± 0.18 cm and 1.40 ± 0.11 cm, and longest-lasting, for more than 120 min, elevation effect on porcine submucosal stomach and colon tissues, respectively. Thus, it can be concluded that polymeric solution MCS05 might be considered as a promising tool for use in human EMR and ESD.

The influence of crossbite in early development of mandibular bone asymmetries in paediatric patients.

BACKGROUND: All authors agree that posterior crossbite is a malocclusion that affects mandibular growth and may lead to skeletal asymmetry but there are few data on which age these modifications are easily quantifiable. MATERIAL AND METHODS: For this study, the researchers used x-ray records of 217 children between 6 and 9 years of age, in the mixed dentition stage and with unilateral posterior crossbite. All the horizontal variables were traced and evaluated by the principal researcher, using the tpsDig version 2 computer program. Subsequently, a descriptive and statistical analysis was carried out, using the SPSS 17.0 for Windows program. RESULTS AND DISCUSSION: After analysing the vertical mandibular traces on the x-rays, the researchers found, in all cases, quantifiable differences between the crossbite side and the non-crossbite side. The differences between horizontal variables were statistically significant (p<0.005) for the entire sample (H3-H4), in the group of boys (H3-H4) and in the 7-year old age group (H1-H2 and H3-H4). Differences were observed in the size of the horizontal measures between the crossbite side and the non-crossbite side. Some of these differences were significant as a function of the sex and age of the study sample. Key words:Crossbite, Mandibular asymmetry, Panoramic.

Methylprednisolone Administration Following Spinal Cord Injury Reduces Aquaporin 4 Expression and Exacerbates Edema.

Spinal cord injury (SCI) is an incapacitating condition that affects motor, sensory, and autonomic functions. Since 1990, the only treatment administered in the acute phase of SCI has been methylprednisolone (MP), a synthetic corticosteroid that has anti-inflammatory effects; however, its efficacy remains controversial. Although MP has been thought to help in the resolution of edema, there are no scientific grounds to support this assertion. Aquaporin 4 (AQP4), the most abundant component of water channels in the CNS, participates in the formation and elimination of edema, but it is not clear whether the modulation of AQP4 expression by MP plays any role in the physiopathology of SCI. We studied the functional expression of AQP4 modulated by MP following SCI in an experimental model in rats along with the associated changes in the permeability of the blood-spinal cord barrier. We analyzed these effects in male and female rats and found that SCI increased AQP4 expression in the spinal cord white matter and that MP diminished such increase to baseline levels. Moreover, MP increased the extravasation of plasma components after SCI and enhanced tissue swelling and edema. Our results lend scientific support to the increasing motion to avoid MP treatment after SCI.

Targeted gene delivery by new folate-polycationic amphiphilic cyclodextrin-DNA nanocomplexes in vitro and in vivo.

AIM: Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). EXPERIMENTAL SECTION: The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (ß-CD) core and 14 hexanoyl chains at the secondary face. RESULTS AND CONCLUSIONS: T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 µg FA/µg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand-DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.

Paralogous ALT1 and ALT2 retention and diversification have generated catalytically active and inactive aminotransferases in Saccharomyces cerevisiae.

BACKGROUND: Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. PRINCIPAL FINDINGS: Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. CONCLUSIONS: ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.

Polycationic amphiphilic cyclodextrin-based nanoparticles for therapeutic gene delivery.

AIM: In this study, a set of polycationic amphiphilic cyclodextrins featuring self-assembling capabilities in the presence of nucleic acids have been evaluated as therapeutic gene vectors for in vivo purposes. MATERIALS & METHODS: A tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups in the primary face of the cyclooligosaccharide core and 14 hexanoyl chains in the secondary face was judged to be optimal for therapeutic gene delivery. RESULTS & CONCLUSION: This compound efficiently mediated serum-resistant transfection in HeLa and HepG2 cells, comparing favorably with branched poly(ethyleneimine), with a low associated toxicity. Further transfection experiments using an encoding therapeutic gene plasmid (pCMVIL-12) were effected to report expression levels of IL-12. Finally, in vivo gene delivery experiments by systemic injection in mice indicated relatively high transfection levels in the liver, overcoming trapping of the nanoparticles in lung cells, with low toxicity.

Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme.

BACKGROUND: Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. PRINCIPAL FINDINGS: Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. CONCLUSIONS: Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism.

Gln3-Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion.

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae.

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator--the Hap2-3-5-Gln3 hybrid modulator--harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently.

Effect of alphas1-casein ( CSN1S1) genotype on milk CSN1S1 content in Malagueña and Murciano-Granadina goats.

There is substantial evidence showing that the polymorphism of the goat alphas1-casein (CSN1S1) gene has a major effect on milk protein, casein and fat content as well as on cheese yield. However, its influence on the synthesis rate of CSN1S1 has been less studied, with measurements only available in French breeds. In this article, we have measured milk CSN1S1 content in 89 Malagueña and 138 Murciano-Granadina goats with 305 and 460 phenotypic registers, respectively. In the Malagueña breed, average values of CSN1S1 content estimated for BB, BF, EE and FF genotypes were 6.94+/-0.38, 5.36+/-0.22, 4.58+/-0.13 and 3.98+/-0.27 g/l, respectively, being all significantly different (P<0.05). Conversely, in the Murciano-Granadina breed only the BB genotype (8.50+/-0.60 g/l) was significantly associated with increased levels of CSN1S1 (P<0.05), whereas BF (6.56+/-0.82 g/l), EE (6.39+/-0.60 g/l) and EF (6.91+/-0.76 g/l) genotypes displayed non-significant differences when compared with each other. Our results highlight the existence of breed-specific genetic and/or environmental factors modulating the impact of the CSN1S1 gene polymorphism on the synthesis rate of the corresponding protein.

[Leprosy: control of household contacts in the municipality of Londrina-PR for a ten-year period]. / Hanseníase: o controle dos contatos no município de Londrina-PR em um período de dez anos.

The purpose of this descriptive study is to analyze variables related to leprosy patients' household contacts who received treatment in Londrina-PR-Brazil for a ten-year period. The data analysis was based on the health service's records and from a system of infectious disease. Out of 1055 leprosy's patients, it was recorded 3394 contacts with an average of 3,2. The most exposed individuals were those aged up to 40 (71,5%); son/daughter (40.6%) and husband/wife (17.8%). Of the1731 contacts (51.0%) examined, 183 showed some signs of the disease: there were 16 confirmed cases, 47 were excluded and 120 did not finish the clinical investigation. Most of the contacts (51.6%) were exposed to the multibacillary forms and 12.8% proved they were vaccinated with two doses of BCG. It is possible to conclude that the follow-up of the contacts was not adequate.

Hanseníase: o controle dos contatos no município de Londrina-PR em um período de dez anos / Lepra: el control de los contactos en el municipio de Londrina-PR en un periodo de diez años / Leprosy: control of household contacts in the municipality of Londrina-PR for a ten-year period

Estudo descritivo, com o objetivo de analisar variáveis relacionadas aos contatos de pacientes com hanseníase atendidos no município de Londrina, num período de dez anos. Os dados foram obtidos das fichas de comunicantes e do SINAN. Dentre os 1055 casos de hanseníase, foram registrados 3394 contatos, com média de 3,2. Os indivíduos mais expostos possuem até 40 anos de idade (71,5 por cento), sendo filhos/a (40,6 por cento) e esposo/a (17,8 por cento). Foram examinados 1731 (51,0 por cento) contatos, dos quais, 183 apresentavam algum sinal de hanseníase: confirmados 16 casos, descartados 47 e não concluíram a investigação 120 (65,6 por cento). A maioria dos contatos (51,6 por cento) foi exposta às formas multibacilares e 10,1 por cento comprovaram a efetivação de duas doses da BCG. Conclui-se que está havendo falhas no acompanhamento dos contatos.

The purpose of this descriptive study is to analyze variables related to leprosy patients' household contacts who received treatment in Londrina-PR-Brazil for a ten-year period. The data analysis was based on the health service's records and from a system of infectious disease. Out of 1055 leprosy's patients, it was recorded 3394 contacts with an average of 3,2. The most exposed individuals were those aged up to 40 (71,5 percent); son/daughter (40.6 percent) and husband/wife (17.8 percent). Of the1731 contacts (51.0 percent) examined, 183 showed some signs of the disease: there were 16 confirmed cases, 47 were excluded and 120 did not finish the clinical investigation. Most of the contacts (51.6 percent) were exposed to the multibacillary forms and 12.8 percent proved they were vaccinated with two doses of BCG. It is possible to conclude that the follow-up of the contacts was not adequate.

Estudio descriptivo, con el objetivo de analizar variables relacionadas a los contactos de pacientes con Lepra que son atendidos en el município de Londrina, en un período de diez años. Los datos fueron obtenidos en las fichas de los acompañantes y del SINAN (órgano del MInisterio de Salud que registra la ocurrencia de toda enfermedad transmisible). Entre los 1055 casos de hanseníase, fueron registrados 3394 contactos, con una media de 3,2. Los individuos más expuestos tienen hasta 40 años de edad (71,5 por ciento), siendo hijos/as (40,6 por ciento) y esposos/as (17,8 por ciento). Fueron examinados 1731 (51,0 por ciento) contactos, de los cuales, 183 presentaban alguna señal de la lepra: confirmados 16 casos, descartados 47 y sin concluir la investigación 120. La mayoría de los contactos (51,6 por ciento) fue expuesta a las formas multibacilares y 12,8 por ciento comprobó la administración de dos dosis de la BCG. Se concluye que está habiendo fallas en el acompañamiento de los contactos.

Specialization of the paralogue LYS21 determines lysine biosynthesis under respiratory metabolism in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the first committed step of the lysine biosynthetic pathway is catalysed by two homocitrate synthases encoded by LYS20 and LYS21. We undertook a study of the duplicate homocitrate synthases to analyse whether their retention and presumable specialization have affected the efficiency of lysine biosynthesis in yeast. Our results show that during growth on ethanol, homocitrate is mainly synthesized through Lys21p, while under fermentative metabolism, Lys20p and Lys21p play redundant roles. Furthermore, results presented in this paper indicate that, in contrast to that which had been found for Lys20p, lysine is a strong allosteric inhibitor of Lys21p (K(i) 0.053 mM), which, in addition, induces positive co-operativity for alpha-ketoglutarate (alpha-KG) binding. Differential lysine inhibition and modulation by alpha-KG of the two isozymes, and the regulation of the intracellular amount of the two isoforms, give rise to an exquisite regulatory system, which balances the rate at which alpha-KG is diverted to lysine biosynthesis or to other metabolic pathways. It can thus be concluded that retention and further biochemical specialization of the LYS20- and LYS21-encoded enzymes with partially overlapping roles contributed to the acquisition of facultative metabolism.

Telomere shortening is associated with poor prognosis and telomerase activity correlates with DNA repair impairment in non-small cell lung cancer.

BACKGROUND AND PURPOSE: Telomere function and DNA damage response pathways are frequently inactivated in cancer. Moreover, some telomere-binding proteins have been implicated in DNA repair. The purpose of this work consists of evaluating the prognostic impact of telomere dysfunction and its relationship with DNA repair systems in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: We analysed 83 NSCLCs and their corresponding control samples obtained from patients submitted to surgery. Telomere function was evaluated by determining telomerase activity and telomere length. DNA repair expression assays were established by using cDNA arrays containing 96 DNA-repair genes and by Real Time Quantitative PCR. RESULTS: Our data indicated that telomere attrition was significantly associated with poor clinical outcome of patients (P=0.02), being this parameter a significant prognostic factor independent of tumour stage (P=0.012; relative risk=1.887; 95% CI: 1.147-3.102). DNA-repair gene expression studies showed down regulation of DCLRE1C and GTF2H1 and a clear FLJ10858 up regulation in tumour tissues, as compared to controls. In addition, a number of genes related to DNA-repair were significantly down regulated in tumours that reactivated telomerase (DCLRE1C, GTF2H1, PARP-3, MLH1, and TRF2). CONCLUSIONS: Telomere shortening emerged as a poor clinical evolution parameter in NSCLC. Moreover, results from this work suggest a relationship between the loss of several DNA repair genes and telomerase activity, which may be of relevance in the pathogenesis of non-small lung cancer.

Expression of MMP-9 and TIMP-1 as prognostic markers in gastric carcinoma.

BACKGROUND/AIMS: The aim of the present work is to clarify the role of metalloproteinase-9 and its inhibitor in the evolution of gastric cancer after surgical resection. METHODOLOGY: We have studied 44 gastric cancer patients submitted to surgery. There were 13 proximal tumors, 16 located in the middle third and 15 in the distal one. Overall survival was 26% at 6 years. Metalloproteinase-9 and tissue inhibitor of metalloproteinase concentrations were investigated by means of ELISA in frozen samples of tumoral and normal gastric mucosa. RESULTS: Mean concentration of metalloproteinase-9 in tumoral tissue was 42 ng/mg of total protein, and this value was 6.9 times greater than the mean concentration in non-tumoral tissue. Cancer tissue also expressed higher levels of TIMP-1, 7.25 versus 4.39 ng/mg of protein. Higher levels of metalloproteinase expression in tumoral tissue, greater [metalloproteinase in tumor]/[metalloproteinase in non-tumor] ratio and greater [metalloproteinase]/[inhibitor] ratio in tumor cells, were all of them statistically related to a worse prognosis when T1 and T2 tumors were considered. CONCLUSIONS: The expression of metalloproteinase-9 or its inhibitor is related to a more aggressive phenotype of gastric cancer.