Developmentally regulated MAPK pathways modulate heterochromatin in Saccharomyces cerevisiae

Variegated expression of genes contributes to phenotypic variation within populations of genetically identical cells. Such variation plays a role in development and host pathogen interaction and can be important in adaptation to harsh environments. The expression state of genes placed near telomeres shows a variegated pattern of inheritance due to heterochromatin formation, a phenomenon that is called telomere position effect (TPE). We show that in budding yeast, TPE is controlled by the a1/α2 developmental repressor, which dictates developmental decisions in response to environmental changes. Two a1/α2 repressed genes, STE5, a MAPK scaffold and HOG1, a stress-activated MAPK, are the targets of this heterochromatin regulation pathway. We provide new evidence that link MAPK signaling and heterochromatin formation in yeast. Our results show that the same components that regulate gene expression states in euchromatic regions regulate heterochromatic expression states and that stress can play a part in turning on or off genes placed in heterochromatic regions.     

IP-FCM: Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.Download video file.^{(71M, mov)}     

Adaptation and Acclimation of Photosynthetic Microorganisms to Permanently Cold Environments

Persistently cold environments constitute one of our world's largest ecosystems, and microorganisms dominate the biomass and metabolic activity in these extreme environments. The stress of low temperatures on life is exacerbated in organisms that rely on photoautrophic production of organic carbon and energy sources. Phototrophic organisms must coordinate temperature-independent reactions of light absorption and photochemistry with temperature-dependent processes of electron transport and utilization of energy sources through growth and metabolism. Despite this conundrum, phototrophic microorganisms thrive in all cold ecosystems described and (together with chemoautrophs) provide the base of autotrophic production in low-temperature food webs. Psychrophilic (organisms with a requirement for low growth temperatures) and psychrotolerant (organisms tolerant of low growth temperatures) photoautotrophs rely on low-temperature acclimative and adaptive strategies that have been described for other low-temperature-adapted heterotrophic organisms, such as cold-active proteins and maintenance of membrane fluidity. In addition, photoautrophic organisms possess other strategies to balance the absorption of light and the transduction of light energy to stored chemical energy products (NADPH and ATP) with downstream consumption of photosynthetically derived energy products at low temperatures. Lastly, differential adaptive and acclimative mechanisms exist in phototrophic microorganisms residing in low-temperature environments that are exposed to constant low-light environments versus high-light- and high-UV-exposed phototrophic assemblages.     

Multidimensional analysis of immune responses identified biomarkers of recent Mycobacterium tuberculosis infection

The risk of tuberculosis (TB) disease is higher in individuals with recent Mycobacterium tuberculosis (M.tb) infection compared to individuals with more remote, established infection. We aimed to define blood-based biomarkers to distinguish between recent and remote infection, which would allow targeting of recently infected individuals for preventive TB treatment. We hypothesized that integration of multiple immune measurements would outperform the diagnostic performance of a single biomarker. Analysis was performed on different components of the immune system, including adaptive and innate responses to mycobacteria, measured on recently and remotely M.tb infected adolescents. The datasets were standardized using variance stabilizing scaling and missing values were imputed using a multiple factor analysis-based approach. For data integration, we compared the performance of a Multiple Tuning Parameter Elastic Net (MTP-EN) to a standard EN model, which was built to the individual adaptive and innate datasets. Biomarkers with non-zero coefficients from the optimal single data EN models were then isolated to build logistic regression models. A decision tree and random forest model were used for statistical confirmation. We found no difference in the predictive performances of the optimal MTP-EN model and the EN model [average area under the receiver operating curve (AUROC) = 0.93]. EN models built to the integrated dataset and the adaptive dataset yielded identically high AUROC values (average AUROC = 0.91), while the innate data EN model performed poorly (average AUROC = 0.62). Results also indicated that integration of adaptive and innate biomarkers did not outperform the adaptive biomarkers alone (Likelihood Ratio Test χ² = 6.09, p = 0.808). From a total of 193 variables, the level of HLA-DR on ESAT6/CFP10-specific Th1 cytokine-expressing CD4 cells was the strongest biomarker for recent M.tb infection. The discriminatory ability of this variable was confirmed in both tree-based models.A single biomarker measuring M.tb-specific T cell activation yielded excellent diagnostic potential to distinguish between recent and remote M.tb infection.     

Amniotic fluid prostaglandin E2 in preterm labor

These studies were designed to determine amniotic fluid concentrations of prostaglandin E2 (PGE) in women with preterm labor. Amniotic fluid was retrieved by transabdominal amniocentesis from 68 women with preterm labor (less than 37 weeks). Patients were divided into three groups according to the response to tocolysis and the presence or absence of an intraamniotic infection. Amniotic fluid concentrations of PGE2 were significantly greater in women with preterm labor and intraamniotic infection than in women without infection. Patients unresponsive to tocolysis without intraamniotic infection had a significantly greater concentration of PGE2 in amniotic fluid than those responsive to tocolysis.     

Amniotic fluid concentration of 5-hydroxyeicosatetraenoic acid is increased in human parturition at term

5-hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if spontaneous human labor at term is associated with changes in the concentration of this metabolite in amniotic fluid. Fluid was retrieved from 36 women not in labor and from 30 women in active labor at term. 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE of women in labor was significantly greater than that of women not in labor (3538 pg/ml vs. 1977 pg/ml, respectively; p = 0.05). This observation is consistent with activation of the lipoxygenase pathway of arachidonic acid metabolism during spontaneous human parturition at term.     

Use of SSCP analysis to identify germline mutations in HNPCC families fulfilling the Amsterdam criteria

Hereditary non-polyposis colorectal cancer (HNPCC) is a clinical syndrome characterised by an inherited predisposition to early onset colorectal and uterine cancers and an increased incidence of other cancers. It is caused by germline defects in the human mismatch repair genes. Defects in two of the known mismatch repair genes (namely hMSH2 and hMLH1) account for over 90% of mutations found in HNPCC families. In this study we have identified 14 families that fulfilled the clinical criteria for HNPCC and screened the hMSH2 and hMLH1 genes for germline mutations using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Seven mutations were identified. Of these, there were five frameshifts, one missense mutation and a further novel mutation that involved separate transition and transversion changes in successive amino acid residues. Three of the mutations were in hMSH2 and four in hMLH1. The identification of germ-line mutations in an HNPCC family enables targeted surveillance and the possibility of early curative intervention. SSCP is a simple and effective method for identifying most mutations in the human mismatch repair genes using DNA from fresh, frozen or archival material. Received: 24 July 1996 / Revised: 26 September 1996     

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such asN-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regeneration may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.     

Molinacuaria indonesiensis n. sp. (Nematoda,Acuarioidea) from Rattus argentiventer in Indonesia

Molinacuaria indonesiensis n. sp. from the stomach of Rattus argentiventer, the ricefield rat, in Sukamandi, Java, Indonesia is described and figured from the examination of seven males and three females. The species is separated from the three other species of the genus, namely, M. bendelli (Adams & Gibson, 1969) Wong & Lankester, 1985, M. acholonui (Schmidt & Kuntz, 1972) Wong & Lankester, 1985 and M. gallinulae (Wang, 1966) Wong & Lankester, 1985. The species is characterised by (i) the larger body measurements, (ii) the presence of a fold rather than a deep groove separating the anterior cephalic region and the ptilina which form four distinct shields, (iii) the morphology of the four ptilina, each one being tripartite with a pointed middle section twice as long at the two lateral sections (iv) the number of papillae on the posterior end of the male (two pairs pre-cloacal, four pairs post-cloacal), (v) the morphology of the left spicule, and (vi) the female tail being straight with a terminal knob.