S100A2 is a BRCA1/p63 coregulated tumour suppressor gene with roles in the regulation of mutant p53 stability

Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

"Things I did not know”: Retrospectives on a Canadian rural male youth suicide using an instrumental photo voice case study

In Canada, it is young rural based men who are at the greatest risk for suicide. While there is no consensus on the reasons for this, evidence points to contextual social factors including isolation, lack of confidential services and pressure to uphold restrictive norms of rural masculinity. In this article we share findings drawn from an instrumental photo voice case study to distil factors contributing to the suicide of a young Canadian rural based man. Integrating photo voice methods and in-depth qualitative we conducted interviews with 7 family members and close friends of the deceased. The interviews and image data were analyzed using constant comparative methods to discern themes related to participants’ reflections on and perceptions about rural male suicide. Three inductively derived themes, “Missing the signs”, “Living up to his public image” and “Down in Rural Canada ” reflect the challenges that survivors and young rural men can experience in attempting to be comply with restrictive dominant ideals of masculinity. We conclude that community based suicide prevention efforts would benefit from gender-sensitive and place specific approaches to advancing men’s mental health by making tangibly available and affirming an array of masculinities to foster the well-being of young rural based men.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action.

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.

Analysis of a temperature sensitive mutation affecting aldehyde oxidase activity in Drosophila melanogaster

A strain of Drosophila melanogaster (mid america stock culture no. hl16) has been reported to be deficient in aldehyde oxidase activity (Hickey and Singh 1982). This strain was characterized during the course of this study and compared to other mutant strains known to be deficient in aldehyde oxidase activity. During the course of this investigation, the hl16 strain was found to be temperature sensitive in its viability. It was found that the two phenotypes, the enzyme deficiency, and the temperature sensitive lethality were the result of two different mutations, both mapping to the X-chromosome. These two mutations were found to be separable by recombination. The enzyme deficiency was found to map to the same locus as the cinnamon mutation, another mutation which affects aldehyde oxidase production. The developmental profile of aldehyde oxidase in the hl16 strain was compared to the developmental profile in the Canton S wild type strain. The aldehyde oxidase activity in adult hl16 individuals was also compared to that of various other strains. It was also found that the aldehyde oxidase activity was temperature sensitive in the adult flies. The temperature sensitive lethality mutation was mapped to position 1-0.1.

Influence of temperature and photoperiod upon ionic regulation in rainbow trout Salmo gairdneri

Interactions of photoperiod and temperature upon waterelectrolyte balance were examined in rainbow trout acclimated to six combinations of two photoperiods {18h light: 6h dark, o 6h light: l8h dark) and three temperatures (2, 10 and 18 C). The influence of temperature and photoperiod upon plasma, skeletal muscle, cardiac muscle and liver levels of sodium, potassium, magnesi.um, calcium, chloride, water content, water distribution and cellular ion concentrations was determined by a one way analysis of variance. Significant (p < 0.05 or better) temperature effects at common photoperiods were observed in 70% of the analyses performed, showing no bias toward either photoperiod. Significant photoperiod effects occured in 57% of the analyses performed at common temperatures. The influence of photoperiod was most prevalent at reduced temperatures. Potassium and magnesium appeared to be particularly thermosensitive, while sodium and calcium were the most photosensitive of the electrolytes. The ionic composition of all tissues studied were relatively thermosensitive, with liver apparently being the most sensitive. On the other hand; the ionic composition of skeletal and cardiac muscle appear to be the mos.t photosensitive of the tissues examined. Water content and distribution in skeletal muscle and liver were significantly influenced by temperature in 50% of the analyses performed showing a very strong bias toward UwinterU animals. Photoperiod effects were significant in 56% of the water parameters measured with a strong bias toward the two lower temperatures. Body weight was of significant influence in 16% of the 174 analyses performed. These data are discussed in terms of the effect of temperature upon ionregulatory mechanisms and the possible impact of photoperiod variations on endocrine systems influencing water-electrolyte metabolism.

Pathogenèse moléculaire de la neuropathie sensitive et motrice héréditaire avec agénésie du corps calleux

La neuropathie sensitive et motrice héréditaire avec agénésie du corps calleux (NSMH/ACC) se traduit par une atteinte neurodégénérative sévère associée à des anomalies développementales dans le système nerveux central et du retard mental. Bien que rare dans le monde, ce désordre autosomique récessif est particulièrement fréquent dans la population Québécoise du Canada Français du fait d’un effet fondateur. L’unique étude réalisée sur la mutation québécoise du gène qui code pour le co-transporteur de potassiumchlore 3 (KCC3) a montré qu’il y a une perte de fonction de la protéine. Cependant, la maladie est également retrouvée hors du Québec et il reste encore à élucider les pathomécanismes mis en jeu. Nous avons donc séquencé les 26 exons du gène KCC3 chez des individus recrutés dans le monde entier et suspectés d’être atteints de la maladie. Nous avons ainsi identifié trois nouvelles mutations. L’étude fonctionnelle de ces mutations nous a confirmé la perte de fonction systématique des co-transporteurs mutés. Puisque l’inactivation de KCC3 se produit majoritairement via l’élimination de segments peptidiques en C-terminus, nous avons concentré notre attention sur l’identification des interactions qui s’y produisent. À l’aide d’approches double hybride, pull-down et immunomarquage, nous avons déterminé que KCC3 interagit avec la créatine kinase CK-B et que cette interaction est perturbée par les mutations tronquantes. De plus, l’utilisation d’un inhibiteur de créatine kinase inactive KCC3, ce qui démontre qu’il existe bien un lien fonctionnel et pathologique entre KCC3 et ses partenaires C-terminaux. Nous avons aussi identifié des anomalies majeures de localisation membranaire des KCC3 mutés. Que KCC3 soit tronqué ou pleine longueur, sa distribution subcellulaire est affectée dans des cellules en culture, dans les ovocytes de Xenopes et dans des échantillons de cerveau de patients. La perte d’interaction entre KCC3 et CK-B et/ou les défauts de transit intracellulaire de KCC3 sont donc les mécanismes pathologiques majeurs de la NSMH/ACC.

Une lésion neurotoxique de l’habenula latérale amplifie la locomotion induite par un psychostimulant sans altérer la récompense

L’habenula, un noyau épithalamique, est située au centre de la voie dorsale diencéphalique. Cette voie relie les structures limbiques et les ganglions de la base aux cellules monoaminergiques du mésencéphale. En particulier, l’habenula latérale (HbL) projette directement aux cellules dopaminergiques et GABAergiques de l’aire tegmentale ventrale (ATV). L’ATV est le site d’origine de la voie mésolimbique dopaminergique, une voie impliquée de façon cruciale dans la manifestation des comportements dirigés. L’importance de cette projection habenulaire pour le comportement demeure encore méconnue. Ainsi, l’objectif de cette étude est d’approfondir notre compréhension du rôle de régulation de l’HbL sur les comportements dépendants de la neurotransmission dopaminergique. MATÉRIEL ET MÉTHODES: Des rats adultes mâles Sprague-Dawley ont été anesthésiés avec de l’isofluorane et installés sur un appareil stéréotaxique. L’acide iboténique, une neurotoxine agoniste des récepteurs glutamatergiques, était infusée bilatéralement dans l’HbL (0,25 μg/0,25 μl/côté). Les rats du groupe contrôle recevaient des infusions NaCl 0,9%. Les rats de l’expérience d’autostimulation intracérébrale (ASIC) étaient aussi implantés d’une électrode monopolaire dans le mésencéphale postérieur. Un groupe de rats était testé pour leur réponse de locomotion à l’amphétamine (0; 0,5 ou 1 mg/kg, intrapéritonéal), dix jours suivant la lésion de l’HbL. La locomotion était mesurée dans des chambres d’activité, chacune équipée de deux faisceaux parallèles infrarouges. Le jour du test, les rats étaient pesés et placés dans la chambre d’activité puis leur activité locomotrice de base était mesurée pendant une heure. Les rats recevaient ensuite une dose d’amphétamine ou le véhicule (NaCl 0,9%) par voie intrapéritonéale et l’activité locomotrice était mesurée pendant deux heures supplémentaires. Un groupe de rats distinct a été utilisé dans l’expérience d’ASIC. Commençant sept jours suivant la lésion, les rats étaient entraînés à appuyer sur un levier afin de s’autoadministrer des stimulations électriques, au cours de sessions quotidiennes. Nous avons ensuite mesuré chacun des taux de réponses d’une série de stimulations aux fréquences décroissantes. À partir d’une courbe réponses-fréquences, le seuil de récompense était inféré par la fréquence de la stimulation nécessaire pour produire une réponse semi-maximale. Les seuils de récompense étaient stabilisés à un niveau similaire pour l’ensemble des rats. Enfin, l’effet sur la récompense de l’amphétamine était testé aux mêmes doses employées pour l’expérience de locomotion. RÉSULTATS: Une lésion neurotoxique de l’HbL n’a pas altéré les niveaux de base de l’activité locomotrice dans chaque groupe. Cependant, une telle lésion a potentialisé l’effet de locomotion de l’amphétamine (1 mg/kg) pendant la première heure suivant son administration, et une tendance similaire était observable pendant la seconde heure. À l’inverse, nous n’avons observé aucune interaction entre une lésion à l’HbL et l’effet amplificateur sur la récompense de l’amphétamine. CONCLUSION: Nos résultats révèlent une importante contribution fonctionnelle de l’HbL à la locomotion induite par l’activation de la voie mésolimbique dopaminergique avec une dose de 1 mg/kg d’amphétamine. À l’opposé, aucun effet sur la récompense n’a été observé. Ces résultats suggèrent que l’activation psychomotrice et l’amplifiation de la récompense produite par l’amphétamine dépendent de substrats dissociables, chacun étant différentiellement sensible à la modulation provenant de l’HbL.

Further characterization and determination of the single amino acid change in the tsI138 reovirus thermosensitive mutant

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the bio- logical properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the l1 protein. In the present study, this mu- tant was further studied as a possible tool to establish the role of the putative l1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

Effect of two opposing changes in photoperiod upon age at first egg in layer-hybrid pullets

An experiment was designed to test the response of growing pullets to two changes in photoperiod (an increase from 8 to 14 h followed 5 weeks later by the reverse change, or a decrease from 14 to 8 h followed by an increase). The first change was made either at 35 days or at 56 days of age, to test the influence of age on the responses observed. Control groups were kept oil constant 8-h and constant 14-h photoperiods and the responses to appropriate single changes were also tested. Mean age at first egg varied from 111 days for birds given a single increment at 56 days to 166 days for pullets given an increase in photoperiod at 35 days followed by a reduction at 70 days. Responses to the single changes confirmed earlier reports that sensitivity to change in photoperiod varies with age ill a manner that is quantitatively predictable. Responses to the double changes could be explained by Postulating that the initial change altered the 'physiological age' of the bird to all extent that was also quantitatively predictable. An early increase in photoperiod advances sexual development and makes the bird more sensitive to a subsequent decrease than would be expected by reference to its chronological age. An early decrease in photoperiod delays sexual development, which can have the effect of making the bird more or less sensitive to a subsequent increase since, ill layer-strain pullets, sensitivity to an increment in photoperiod normally increases Lip to about 9 weeks of age but decreases thereafter. Mean age at first egg predicted using these concepts was very highly correlated with observed age at first egg. The results provide a rational basis for constructing a model to predict age at first egg for any combination of increases and decreases in photoperiod applied to growing pullets.

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 degreesC, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. C-w was lower). The low-moisture-content limit (m(c)) to this relation also differed, being lower in the mutant near-isogenic lines (5.4-5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semilogarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity. was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below mc where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 °C, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. CW was lower). The low-moisture-content limit (mc) to this relation also differed, being lower in the mutant near-isogenic lines (5.4–5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semi-logarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below mc where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.

The variant rpoS allele of S-enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::str(r) null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::str(r). Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.