Sourdough bread enriched with exopolysaccharides and gazpacho by-products modulates in vitro the microbiota dysbiosis.

Sourdough bread enriched with soluble fiber (by in-situ exopolysaccharides production) and insoluble fiber (by gazpacho by-products addition) showed prebiotic effects an in vitro dynamic colonic fermentation performance with obese volunteer's microbiota. Bifidobacterium population was maintained whereas Lactobacillus increased throughout the colonic sections. Conversely, Enterobacteriaceae and Clostridium groups clearly decreased. Specific bacteria associated with beneficial effects increased in the ascending colon (Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium longum and Bifidobacterium adolescentis) whereas Eubacterium eligens, Alistipes senegalensis, Prevotella copri and Eubacterium desmolans increased in the transversal and descending colon. Additionally, Blautia faecis and Ruminococcus albus increased in the transversal colon, and Bifidobacterium longum, Roseburia faecis and Victivallis vadensis in the descending colon. Bifidobacterium and Lactobacillus fermented the in-situ exopolysaccharides and released pectins from gazpacho by-products, as well as cellulosic degraded bacteria. This increased the short and medium chain fatty acids. Acetic acid, as well as butyric acid, increased throughout the colonic tract, which showed greater increases only in the transversal and descending colonic segments. Conversely, propionic acid was slightly affected by the colonic fermentation. These results show that sourdough bread is a useful food matrix for the enrichment of vegetable by-products (or other fibers) in order to formulate products with microbiota modulatory capacities.

Fiber from elicited butternut pumpkin (Cucurbita moschata D. cv. Ariel) modulates the human intestinal microbiota dysbiosis.

Elicited pumpkin was evaluated as a potential daily consumption product able to modulate the gut microbiota. An in vitro dynamic colonic fermentation performance with microbiota from obese volunteers was used. Prebiotic effects were observed after the pumpkin treatment. Bifidobacterium abundance was maintained during the treatment period whereas Lactobacillus increased in the transversal and descending colon. Conversely, Enterobacteriaceae and Clostridium groups were more stable, although scarce decreasing trends were observed for same species. Increments of Lactobacillus acidophilus and Limosilactobacillus fermentum (old Lactobacillus fermentum) were observed in the whole colonic tract after the treatment period. However, modulatory effects were mainly observed in the transversal and descending colon. Diverse bacteria species were increased, such as Akkermansia muciniphila, Bacteroides dorei, Cloacibacillus porcorum, Clostridium lactatifermentans, Ruminococcus albus, Ruminococcus lactaris, Coprococcus catus, Alistipes shahii or Bacteroides vulgatus. The prebiotic effect of the elicited pumpkin was provided by the fiber of the pumpkin, suggesting a release of pectin molecules in the transversal and distal colonic tract through low cellulosic fiber degradation, explaining the increases in the total propionic and butyric acid in these colonic sections. Also, a possible modulatory role of carotenoids from the sample was suggested since carotenes were found in the descending colon. Hence, the results of this research highlighted pumpkin as a natural product able to modulate the microbiota towards a healthier profile.

Effects of Glucosinolate-Enriched Red Radish (Raphanus sativus) on In Vitro Models of Intestinal Microbiota and Metabolic Syndrome-Related Functionalities.

The gut microbiota profile is determined by diet composition, and therefore this interaction is crucial for promoting specific bacterial growth and enhancing the health status. Red radish (Raphanus sativusL.) contains several secondary plant metabolites that can exert a protective effect on human health. Recent studies have shown that radish leaves have a higher content of major nutrients, minerals, and fiber than roots, and they have garnered attention as a healthy food or supplement. Therefore, the consumption of the whole plant should be considered, as its nutritional value may be of greater interest. The aim of this work is to evaluate the effects of glucosinolate (GSL)-enriched radish with elicitors on the intestinal microbiota and metabolic syndrome-related functionalities by using an in vitro dynamic gastrointestinal system and several cellular models developed to study the GSL impact on different health indicators such as blood pressure, cholesterol metabolism, insulin resistance, adipogenesis, and reactive oxygen species (ROS). The treatment with red radish had an influence on short-chain fatty acids (SCFA) production, especially on acetic and propionic acid and many butyrate-producing bacteria, suggesting that consumption of the entire red radish plant (leaves and roots) could modify the human gut microbiota profile toward a healthier one. The evaluation of the metabolic syndrome-related functionalities showed a significant decrease in the gene expression of endothelin, interleukin IL-6, and cholesterol transporter-associated biomarkers (ABCA1 and ABCG5), suggesting an improvement of three risk factors associated with metabolic syndrome. The results support the idea that the use of elicitors on red radish crops and its further consumption (the entire plant) may contribute to improving the general health status and gut microbiota profile.

Association of the Gut Microbiota with the Host's Health through an Analysis of Biochemical Markers, Dietary Estimation, and Microbial Composition.

This study aims to analyze the relationship between gut microbiota composition and health parameters through specific biochemical markers and food consumption patterns in the Spanish population. This research includes 60 Spanish adults aged 47.3 ± 11.2 years old. Biochemical and anthropometric measurements, and a self-referred dietary survey (food frequency questionnaire), were analyzed and compared with the participant´s gut microbiota composition analyzed by 16s rDNA sequencing. Several bacterial strains differed significantly with the biochemical markers analyzed, suggesting an involvement in the participant´s metabolic health. Lower levels of Lactobacillaceae and Oscillospiraceae and an increase in Pasteurellaceae, Phascolarctobacterium, and Haemophilus were observed in individuals with higher AST levels. Higher levels of the Christensenellaceae and a decrease in Peptococcaceae were associated with higher levels of HDL-c. High levels of Phascolarctobacterium and Peptococcus and low levels of Butyricicoccus were found in individuals with higher insulin levels. This study also identified associations between bacteria and specific food groups, such as an increase in lactic acid bacteria with the consumption of fermented dairy products or an increase in Verrucomicrobiaceae with the consumption of olive oil. In conclusion, this study reinforces the idea that specific food groups can favorably modulate gut microbiota composition and have an impact on host´s health.

An In Vitro Protocol to Study the Modulatory Effects of a Food or Biocompound on Human Gut Microbiome and Metabolome.

The gut microbiota plays a key role in gastrointestinal immune and metabolic functions and is influenced by dietary composition. An in vitro protocol simulating the physiological conditions of the digestive system helps to study the effects of foods/biocompounds on gut microbiome and metabolome. The Dynamic-Colonic Gastrointestinal Digester consists of five interconnected compartments, double jacket vessels that simulate the physiological conditions of the stomach, the small intestine and the three colonic sections, which are the ascending colon, transverse colon and descending colon. Human faeces are required to reproduce the conditions and culture medium of the human colon, allowing the growth of the intestinal microbiota. After a stabilization period of 12 days, a food/biocompound can be introduced to study its modulatory effects during the next 14 days (treatment period). At the end of the stabilization and treatment period, samples taken from the colon compartments are analysed. The 16S rRNA gene analysis reveals the microbiota composition. The untargeted metabolomics analysis gives more than 10,000 features (metabolites/compounds). The present protocol allows in vitro testing of the modulatory effects of foods or biocompounds on gut microbiota composition and metabolic activity.

Gut Microbiota Bacterial Species Associated with Mediterranean Diet-Related Food Groups in a Northern Spanish Population.

The MD (Mediterranean diet) is recognized as one of the healthiest diets worldwide and is associated with the prevention of cardiovascular and metabolic diseases. Dietary habits are considered one of the strongest modulators of gut microbiota, which seem to play a significant role in health status of the host. The purpose of the present study was to evaluate interactive associations between gut microbiota composition and habitual dietary intake in 360 Spanish adults from the Obekit cohort (normal weight, overweight, and obese participants). Dietary intake and adherence to the MD tests were administered and fecal samples were collected from each participant. Fecal 16S rRNA (ribosomal Ribonucleic Acid) gene sequencing was performed and checked against the dietary habits. MetagenomeSeq was the statistical tool applied to analyze data at the species taxonomic level. Results from this study identified several beneficial bacteria that were more abundant in the individuals with higher adherence to the MD. Bifidobacterium animalis was the species with the strongest association with the MD. Some SCFA (Short Chain Fatty Acids) -producing bacteria were also associated with MD. In conclusion, this study showed that MD, fiber, legumes, vegetable, fruit, and nut intake are associated with an increase in butyrate-producing taxa such as Roseburia faecis, Ruminococcus bromii, and Oscillospira (Flavonifractor) plautii.

Genetic identification of a war-evacuated child in search of his own identity for more than seventy years.

V. M. E. was evacuated when he was a young boy in 1939. He left an aunt and cousins in Spain (G. E. family). He was adopted in Belgium by the D. family and thus his new name became V. D. He has been unable to remember his childhood before his adoption, a symptomatology compatible with amnesia for personal identity, presumably because he may have suffered a head contusion before or during his exodus. Identification tests were performed on blood samples from V. D. and V. G. E., a mitochondrial cousin of the missing boy. V. G. E. and the missing boy have a common mitochondrial ancestor, their maternal grandmother. The mitochondrial profile of both samples turned out to be highly specific, which allowed the genetic identification of V. D. as V. M. E. As a result, V. D. has reclaimed his past and reunited with his former family in Spain after more than seven decades. As far as we know, this is the first report describing the application of mitochondrial DNA in the identification of a person evacuated during the Spanish Civil War suffering from amnesia for personal identity.

The Saccharomyces cerevisiae Ptc1 protein phosphatase attenuates G2-M cell cycle blockage caused by activation of the cell wall integrity pathway.

Lack of the yeast Ptc1 Ser/Thr protein phosphatase results in numerous phenotypic defects. A parallel search for high-copy number suppressors of three of these phenotypes (sensitivity to Calcofluor White, rapamycin and alkaline pH), allowed the isolation of 25 suppressor genes, which could be assigned to three main functional categories: maintenance of cell wall integrity (CWI), vacuolar function and protein sorting, and cell cycle regulation. The characterization of these genetic interactions strengthens the relevant role of Ptc1 in downregulating the Slt2-mediated CWI pathway. We show that under stress conditions activating the CWI pathway the ptc1 mutant displays hyperphosphorylated Cdc28 kinase and that these cells accumulate with duplicated DNA content, indicative of a G2-M arrest. Clb2-associated Cdc28 activity was also reduced in ptc1 cells. These alterations are attenuated by mutation of the MKK1 gene, encoding a MAP kinase kinase upstream Slt2. Therefore, our data show that Ptc1 is required for proper G2-M cell cycle transition after activation of the CWI pathway.

Improving Ribosomal RNA Integrity in Surgically Resected Human Brain Tumor Biopsies.

BACKGROUND: Biopsies extracted from brain cancer patients often display degraded ribosomal RNA, which makes them unusable in transcriptomic experiments. This has not been properly documented in previous works aimed at refining the molecular classification of brain cancer. OBJECTIVE: To determine RNA integrity in a large cohort of human brain cancer biopsies and to evaluate different factors that may influence RNA integrity in both a murine model of glioblastoma and in additional subsets of patient biopsies. METHODS: Total RNA was isolated from 255 biopsies of various human brain tumors (HBTs) and processed on a Bioanalyzer. Correct RNA integrity was considered for samples showing either the ribosomal 28S/18S peak ratio ≥ 1.2 or RNA integrity number ≥ 6. The time-dependent effect of ex vivo ischemia was evaluated in a murine model, whose results were tested in a new collection of 27 human biopsies. Multiple biopsy sampling was considered in a further set comprising 32 biopsies. RESULTS: The 255 human biopsies revealed a substantial percentage of samples displaying degraded RNA (27.5%). The murine model confirmed the known relevance of ex vivo ischemia time in increased RNA degradation. Human biopsies extracted immediately after cauterization showed a trend toward less RNA degradation. Combining snap freezing and multiple sampling of biopsies, the percentage of patients with degraded RNA was reduced by twofold (15.6%). CONCLUSIONS: We provide a first concise study of factors influencing RNA degradation in HBT biopsies. Immediate biopsy removal after cauterization of the tumor area, snap freezing, and multiple sampling improve RNA quality.

Robustness of equations that define molecular subtypes of glioblastoma tumors based on five transcripts measured by RT-PCR.

Glioblastoma (Gb) is one of the most deadly tumors. Its molecular subtypes are yet to be fully characterized while the attendant efforts for personalized medicine need to be intensified in relation to glioblastoma diagnosis, treatment, and prognosis. Several molecular signatures based on gene expression microarrays were reported, but the use of microarrays for routine clinical practice is challenged by attendant economic costs. Several authors have proposed discriminant equations based on RT-PCR. Still, the discriminant threshold is often incompletely described, which makes proper validation difficult. In a previous work, we have reported two Gb subtypes based on the expression levels of four genes: CHI3L1, LDHA, LGALS1, and IGFBP3. One Gb subtype presented with low expression of the four genes mentioned, and of MGMT in a large portion of the patients (with anticipated high methylation of its promoter), and mutated IDH1. Here, we evaluate the robustness of the equations fitted with these genes using RT-PCR values in a set of 64 cases and importantly, define an unequivocal discriminant threshold with a view to prognostic implications. We developed two approaches to generate the discriminant equations: 1) using the expression level of the four genes mentioned above, and 2) using those genes displaying the highest correlation with survival among the aforementioned four ones, plus MGMT, as an attempt to further reduce the number of genes. The ease of equations' applicability, reduction in cost for raw data, and robustness in terms of resampling-based classification accuracy warrant further evaluation of these equations to discern Gb tumor biopsy heterogeneity at molecular level, diagnose potential malignancy, and prognosis of individual patients with glioblastomas.

Strategies for annotation and curation of translational databases: the eTUMOUR project.

The eTUMOUR (eT) multi-centre project gathered in vivo and ex vivo magnetic resonance (MR) data, as well as transcriptomic and clinical information from brain tumour patients, with the purpose of improving the diagnostic and prognostic evaluation of future patients. In order to carry this out, among other work, a database--the eTDB--was developed. In addition to complex permission rules and software and management quality control (QC), it was necessary to develop anonymization, processing and data visualization tools for the data uploaded. It was also necessary to develop sophisticated curation strategies that involved on one hand, dedicated fields for QC-generated meta-data and specialized queries and global permissions for senior curators and on the other, to establish a set of metrics to quantify its contents. The indispensable dataset (ID), completeness and pairedness indices were set. The database contains 1317 cases created as a result of the eT project and 304 from a previous project, INTERPRET. The number of cases fulfilling the ID was 656. Completeness and pairedness were heterogeneous, depending on the data type involved.

Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy.

Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.

Development of a predictor for human brain tumors based on gene expression values obtained from two types of microarray technologies.

Development of molecular diagnostics that can reliably differentiate amongst different subtypes of brain tumors is an important unmet clinical need in postgenomics medicine and clinical oncology. A simple linear formula derived from gene expression values of four genes (GFAP, PTPRZ1, GPM6B, and PRELP) measured from cDNA microarrays (n = 35) have distinguished glioblastoma and meningioma cases in a previous study. We herein extend this work further and report that the above predictor formula showed its robustness when applied to Affymetrix microarray data acquired prospectively in our laboratory (n = 80) as well as publicly available data (n = 98). Importantly, GFAP and GPM6B were both retained as being significant in the predictive model upon using the Affymetrix data obtained in our laboratory, whereas the other two predictor genes were SFRP2 and SLC6A2. These results collectively indicate the importance of the expression values of GFAP and GPM6B genes sampled from the two types of microarray technologies tested. The high prediction accuracy obtained in these instances demonstrates the robustness of the predictors across microarray platforms used. This result would require further validation with a larger population of meningioma and glioblastoma cases. At any rate, this study paves the way for further application of gene signatures to more stringent biopsy discrimination challenges.

Automated brain tumor biopsy prediction using single-labeling cDNA microarrays-based gene expression profiling.

AIMS: Gene signatures obtained from microarray experiments may be of use to improve the prediction of brain tumor diagnosis. Nevertheless, automated and objective prediction with accuracy comparable to or better than the gold standard should be convincingly demonstrated for possible clinician uptake of the new methodology. Herewith, we demonstrate that primary brain tumor types can be discriminated using microarray data in an automated and objective way. METHODS: Postsurgical biopsies from 35 patients [17 glioblastoma multiforme (Gbm) and 18 meningothelial meningioma (Mm)] were stored in liquid nitrogen, total RNA was extracted, and cDNA was labeled with Cy3 fluorochrome and hybridized onto a cDNA-based microarray containing 11,500 cDNA clones representing 9300 loci. Scanned data were preprocessed, normalized, and used for predictor development. The predictive functions were fitted to a subset of samples and their performance evaluated with an independent subset. Expression results were validated by means of real time-polymerase chain reaction. RESULTS: Some gene expression-based predictors achieved 100% accuracy both in training resampling validation and independent testing. One of them, composed of GFAP, PTPRZ1, GPM6B and PRELP, produced a 100% prediction accuracy for both training and independent test datasets. Furthermore, the gene signatures obtained, increased cell detoxification, motility and intracellular transport in Gbm, and increased cell adhesion and cytochrome-family genes in Mm, agree well with the expected biologic and pathologic characteristics of the studied tumors. CONCLUSIONS: The ability of gene signatures to automate prediction of brain tumors through a fully objective approach has been demonstrated. A comparison of gene expression profiles between Gbm and Mm may provide additional clues about patterns associated with each tumor type.

Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains.

Haemophilus parasuis is the agent responsible for causing Glässer's disease, but little is known about the pathogenic determinants of this major pig disease. Here we describe, for the pathogenic strain Nagasaki, the molecular characterization of 13 trimeric autotransporters as assessed by the presence of YadA C-terminal translocator domains which were classified into three groups. All passenger domains possess motifs and repeats characteristic of adhesins, hemagglutinins, and invasins with various centrally located copies of collagen-like repeats. This domain architecture is shared with two trimeric autotransporter proteins of H. somnus 129Pt. Genomic comparison by microarray hybridization demonstrated homologies among H. parasuis virulent strains and high divergence with respect to nonvirulent strains. Therefore, these genes were named vtaA (virulence-associated trimeric autotransporters). The sequencing of 17 homologous vtaA genes of different invasive strains highlighted an extensive mosaic structure. Based also on the presence of DNA uptake signal sequences within the vtaA genes, we propose a mechanism of evolution by which gene duplication and the accumulation of mutations and recombinations, plus the lateral gene transfer of the passenger domain, led to the diversity of this multigene family. This study provides insights to help understand the tissue colonization and invasiveness characteristic of H. parasuis pathogenic strains.

A new set of DNA macrochips for the yeast Saccharomyces cerevisiae: features and uses.

The yeast Saccharomyces cerevisiae has been widely used for the implementation of DNA chip technologies. For this reason and due to the extensive use of this organism for basic and applied studies, yeast DNA chips are being used by many laboratories for expression or genomic analyses. While membrane arrays (macroarrays) offer several advantages, for many laboratories they are not affordable. Here we report that a cluster of four Spanish molecular-biology yeast laboratories, with relatively small budgets, have developed a complete set of probes for the genome of S. cerevisiae. These have been used to produce a new type of macroarray on a nylon surface. The macroarrays have been evaluated and protocols for their use have been optimized.

A new set of DNA macrochips for the yeast Saccharomyces cerevisiae: features and uses / Nuevos macrochips para el genoma completo de la levadura Saccharomyces cerevisiae: características y usos

The yeast Saccharomyces cerevisiae has been widely used for the implementation of DNA chip technologies. For this reason and due to the extensive use of this organism for basic and applied studies, yeast DNA chips are being used by many laboratories for expression or genomic analyses. While membrane arrays (macroarrays) offer several advantages, for many laboratories they are not affordable. Here we report that a cluster of four Spanish molecular-biology yeast laboratories, with relatively small budgets, have developed a complete set of probes for the genome of S. cerevisiae. These have been used to produce a new type of macroarray on a nylon surface. The macroarrays have been evaluated and protocols for their use have been optimized (AU)

La levadura Saccharomyces cerevisiae ha sido muy utilizada para el desarrollo de las tecnologías de chips de DNA. Por ese motivo, y porque es un organismo muy utilizado en investigación básica y aplicada, hay muchos laboratorios que usan chips de DNA para estudios genómicos o de expresión. Aunque el uso de macrochips en membrana presenta varias ventajas, su precio los pone fuera del alcance de muchos laboratorios. Aquí mostramos que un grupo de cuatro laboratorios españoles de biología molecular de levaduras ha desarrollado, con presupuestos relativamente bajos, un lote completo de sondas del genoma de S. cerevisiae, que se han usado para fabricar un nuevo tipo de macrochips sobre superficie de nailon. Se han evaluado estos macrochips y se han optimizado los protocolos para su uso (AU)

Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.

Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study.

Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.

Mutagenesis analysis of the yeast Nha1 Na+/H+ antiporter carboxy-terminal tail reveals residues required for function in cell cycle.

The yeast Nha1 Na(+),K(+)/H(+) antiporter may play an important role in regulation of cell cycle, as high-copy expression of the NHA1 gene is able to rescue the blockage at the G(1)/S transition of cells lacking Sit4 protein phosphatase and Hal3 activities. Interestingly, this function was independent of the role of the antiporter in improving tolerance to sodium cations, it required the integrity of a relatively large region (from residues 800 to 948) of its carboxy-terminal moiety, and was not performed by the fission yeast homolog antiporter Sod2, which lacks a carboxy-terminal tail. Here we show that a hybrid protein composed of the Sod2 antiporter fused to the carboxy-terminal half of Nha1 strongly increased sodium tolerance, but did not allow growth at high potassium nor did rescue growth of the sit4 hal3 conditional mutant strain. Deletion of Nha1 residues from 800 to 849, 900 to 925 or 926 to 954 abolished the function of Nha1 in cell cycle without affecting sodium tolerance. A screening for loss-of-function mutations at the 775-980 carboxy-terminal tail of Nha1 has revealed a number of residues required for function in cell cycle, most of them clustering in two regions, from residues 869 to 876 (cluster A) and 918 to 927 (cluster B). The later is rather conserved in other related antiporters, while the former is not.