Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Design of improved membrane protein production experiments: quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightly-defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.     

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen.

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.     

Development of a diploid cell line from fetal rhesus monkey lung for virus vaccine production

Summary The primary goal of this study was to develop and characterize diploid cell lines from fetal tissues of subhuman primates for use in virus vaccine production. Cell lines have been established from fetal tissues of rhesus and African green monkeys, and these have been characterized according to the general criteria recommended by the International Cell Committee for Microbiological Standardization. Of these cell lines, DBS-FRhL-2, developed from lung tissue of a rhesus monkey fetus, has been found to meet the requirements of populations proposed as substrates for virus vaccines. Characterization studies show that DBS-FRhL-2 cells have a finite life of more than 50 population doublings in vitro and maintain the diploid karyotype through an active growth phase. The cells are nontumorigenic, and tests have not revealed the presence of adventitious agents. They are susceptible to a number of human viruses and can be preserved by freezing without change in virus susceptibility, cytogenetic, or growth characteristics. These results indicate the need for further evaluation of this rhesus monkey diploid cell line for acceptability as an alternate substrate in the manufacture of human virus vaccines. The research upon which this publication is based was performed pursuant to Contract No. NIH-69-100 with the Division of Biologics Standards of the National Institutes of Health.     

The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapy that selectively targets cancer cell death while non-malignant cells remain viable. Using a panel of normal human fibroblasts, we characterized molecular differences in human foreskin fibroblasts and WI-38 TRAIL-resistant cells and marginally sensitive MRC-5 cells compared with TRAIL-sensitive human lung and colon cancer cells. We identified decreased caspase-8 protein expression and protein stability in normal fibroblasts compared with cancer cells. Additionally, normal fibroblasts had incomplete TRAIL-induced caspase-8 activation compared with cancer cells. We found that normal fibroblasts lack the ubiquitin modification of caspase-8 required for complete caspase-8 activation. Treatment with the deubiquitinase inhibitor PR-619 increased caspase-8 ubiquitination and caspase-8 enzymatic activity and sensitized normal fibroblasts to TRAIL-mediated apoptosis. Therefore, posttranslational regulation of caspase-8 confers resistance to TRAIL-induced cell death in normal cells through blockade of initiation of the extrinsic cell death pathway.     

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A₁ receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A₁ and A_2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A₁ receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A₁ and A_2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A₁ or A_2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A₁ or A_2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A₁ receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A₁ receptor with a K_i value of 2.06 µM and (2) adenosine A₁ receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A₁ receptor antagonists for the treatment of neurodegenerative disorders.