Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi

In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of ΔmepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In ΔmepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as ΔmepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the ΔmepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi.     

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.     

Independent Association of Hip Circumference with Metabolic Profile in Different Ethnic Groups

Objective: In whites, a larger hip circumference has been shown to be associated with a better metabolic profile, after adjustment for BMI and waist circumference. Our aim was to investigate this association in a variety of ethnic groups, some highly susceptible to type 2 diabetes. Research Methods and Procedures: We measured weight, height, waist and hip circumferences, systolic and diastolic blood pressure, fasting and 2‐hour postload glucose, triglycerides, and high‐density lipoprotein‐cholesterol in 1020 Melanesians, 767 Micronesians, 3697 Indians, and 2710 Creoles from Pacific and Indian Ocean islands. Leptin and body fat percentage were determined in Indian and Creole Mauritians only. Results: In all ethnic groups, larger hip circumference was associated with lower glucose and triglyceride levels in both sexes and higher high‐density lipoprotein levels in women only, after adjustment for waist circumference, BMI, and age. Adjustment for height or leptin did not materially change the results. Discussion: In conclusion, we confirmed the protective association of relatively larger hips in four nonwhite ethnic groups. Leptin does not seem to play a mediating role in this association.     

The new anticancer drug [(2R )-aminomethylpyrrolidine](1,1-cyclobutanedicarboxylato)platinum(II) and its toxic S enantiomer do interact differently with nucleic acids

 The interaction of the two chiral isomers of the new anticancer agent [Pt(ampyr)(cbdca)] (ampyr=aminomethylpyrrolidine, cbdca=cyclobutanedicarboxylate) with 5′-GMP and with short G-containing oligonucleotides has been studied using ¹H and ³¹P NMR, UV-vis spectroscopy and molecular modelling. Each isomer loses the cbdca ligand upon binding to the DNA fragments. Two geometrical isomers of the DNA adducts are formed owing to the presence of the unsymmetric ampyr ligand. These isomers prove to be GG-N7,N7 chelates for d(GpG), d(pGpG) and d(CpGpG). A slight preference for the formation of one geometrical isomer is found in the case of DNA fragments having a phosphate moiety and/or a C base at the 5′-site of the GG sequence. H-bonding interactions from the NH₂ moiety towards the 5′-phosphate group and/or the O atom of the C base clearly favour the formation of one geometrical isomer. The presence of these H-bonds, together with the bulky pyrrolidine ring, has resulted in the unique observation (by ¹H NMR) of NH protons of coordinated amines that do not rapidly exchange in a 99.95% D₂O solution. Temperature-dependence studies show an extremely slow stack ⇄ destack conformational change for the CGG adducts of the S isomer, which could be related to these stable H-bonds of the amine protons towards the oligonucleotide. For the R isomer this stack ⇄ destack conformational change is faster, probably owing to more steric hindrance of the pyrrolidine ring as deduced from the NOESY data, and as also suggested by molecular modelling. The observation of extremely slow rotation around the Pt-N7 bond for [Pt(R-ampyr)(GMP-N7)₂] provides further evidence for increased steric hindrance of the R isomer compared to the S isomer. The rate of binding of the drug to G bases proved to be second order for both isomers; in fact the (toxic) S isomer is about two times more reactive than the (non-toxic) R isomer, as seen from k ₂ values of 0.17±0.01 M^–1s^–1 for [Pt(S-ampyr)(cbdca)] and 0.09±0.01 M^–1s^–1 for [Pt(R-ampyr)(cbdca)]. No solvent-assisted pathway is involved in these reactions, since the complexes prove to be stable in solution for weeks and therefore only a direct attack of the G base on the Pt must be involved. Because hardly any intermediate species can be detected during the reaction, coordination of the second G base must occur much faster than the binding of the first G base. Since direct attack of the nucleobases takes place, steric interactions become extremely important and therefore are likely to determine the reactivity, activity and even the toxicity of such Pt complexes. Received: 12 January 1999 / Accepted: 17 June 1999     

Translation initiation factor 4E inhibits differentiation of erythroid progenitors

Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.     

A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement

The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.     

Mating system and genetic variation in the endangered New Zealand takahe

The takahe (Porphyrio hochstetteri) is ahighly endangered flightless rail that isendemic to New Zealand. Only one remnantpopulation of takahe (120 adults) is left inthe wild in Fiordland, which has been thesource for introductions to four predator-freeislands. The objective of the present studywas to determine the mating system andamount of genetic variation in takahe usingmultilocus DNA profiling, in order to assist inthe management of the island populations. There was no evidence of extra-pair paternityfor the 27 (73%, n = 37) offspring towhich paternity could be resolved. Thepaternity of the remaining 10 offspring couldnot be resolved due to low levels ofminisatellite DNA variation, but in none wasthe resident male excluded. Overall, the DNAresults along with behavioral and life historyinformation indicate that extra-pairfertilizations should be rare or absent, andtakahe join a small but growing list oflong-lived species of birds that have beenshown to exhibit genetic monogamy. Inaddition, the levels of minisatellite DNAvariation detected in takahe are low relativeto those reported for most other known outbredavian populations, and are consistent with theevidence of the takahe's persistence as asmall, isolated population in Fiordland over atleast the last 100 years. The low geneticvariation is discussed in relation to possibleevidence of environment depended inbreedingdepression in translocated island populationsof takahe.