Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.     

HTLV-I protease cleavage of P19/24 substrates is not dependent on NaCl concentration

Understanding the factors that affect the activity of Human T-cell Leukemia Virus type I (HTLV-I) protease is essential for the discovery of inhibitors to be used for the treatment of HTLV-I infection, but little has been reported on the protease to date. Here we report the production of HTLV-I protease in purified yields greater than 150 mg/L, determination of its extinction coefficient, and determination of the optimum conditions for cleavage of the p19/24 substrates (DABCYL)-(GABA)-PQVL-Nph-VMH-(EDANS), (DABSYL)-(GABA)-PQVL-Nph-VMH-(EDANS), and (DABSYL)-(GABA)-PQVLPVMH-(EDANS). The highest activity was found at pH 5.2-5.3 and 37 degrees C. There was no effect on activity upon change in sodium chloride concentration from 0 to 1500 mM. The values of K(m) and k(cat) for cleavage of these substrates by the protease with and without the histidine tag were determined.     

An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Current thoughts on the phosphatidylinositol transfer protein family

Monomeric transport of lipids is carried out by a class of proteins that can shield a lipid from the aqueous environment by binding the lipid in a hydrophobic cavity. One such group of proteins is the phosphatidylinositol transfer proteins (PITP) that can bind phosphatidylinositol and phosphatidylcholine and transfer them from one membrane compartment to another. PITPs are found in both unicellular and multicellular organisms but not bacteria. In mice and humans, the PITP domain responsible for lipid transfer is found in five proteins, which can be classified into two classes based on sequence. Class I PITPs comprises two family members, alpha and beta, small 35 kDa proteins with a single PITP domain which are ubiquitously expressed. Class IIA PITPs (RdgBalphaI and II) are larger proteins possessing additional domains that target the protein to membranes and are only able to bind lipids but not mediate transfer. Finally, Class IIB PITP (RdgBbeta) is similar to Class I in size (38 kDa) and is also ubiquitously expressed. Class III PITPs, exemplified by the Sec14p family, are found in yeast and plants but are unrelated in sequence and structure to Class I and Class II PITPs. In this review we discuss whether PITP proteins are passive transporters or are regulated proteins that are able to couple their transport and binding properties to specific biological functions including inositol lipid signalling and membrane turnover.     

A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.     

Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.     

EVOLUTION OF MICROALGAE IN HIGHLY STRESSING ENVIRONMENTS: AN EXPERIMENTAL MODEL ANALYZING THE RAPID ADAPTATION OF DICTYOSPHAERIUM CHLORELLOIDES (CHLOROPHYCEAE) FROM SENSITIVITY TO RESISTANCE AGAINST 2,4,6‐TRINITROTOLUENE BY RARE PRESELECTIVE MUTATIONS1

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6‐trinitrotoluene (TNT). When algal cultures were exposed to 30 mg·L ^{? 1} 1 Received 9 July 2001. Accepted 23 July 2002.
TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT‐resistant variants that appear after the massive lysis of the TNT‐sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT‐resistant cells; rather it was found that TNT‐resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 × 10 ^{? 5} mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT‐resistant mutants was significantly lower than that of wild‐type cells. Competition experiments between resistant mutants and wild‐type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT‐resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.