Faculty Bibliography

Lipid phosphates can act as signaling molecules to influence cell division, apoptosis, and migration. wunen and wunen2 encode Drosophila lipid phosphate phosphohydrolases, integral membrane enzymes that dephosphorylate extracellular lipid phosphates. wun and wun2 act redundantly in somatic tissues to repel migrating germ cells, although the mechanism by which germ cells respond is unclear. Here, we report that wun2 also functions in germ cells, enabling them to perceive the wun/wun2-related signal from the soma. Upon Wun2 expression, cultured insect cells dephosphorylate and internalize exogenously supplied lipid phosphate. We propose that Drosophila germ cell migration and survival are controlled by competition for hydrolysis of a lipid phosphate between germ cells and soma

BACKGROUND: Human telomeres are coated by the telomere repeat binding proteins TRF1 and TRF2, which are believed to function independently to regulate telomere length and protect chromosome ends, respectively. RESULTS: Here, we show that TRF1 and TRF2 are linked via TIN2, a previously identified TRF1-interacting protein, and its novel binding partner TINT1. TINT1 localized to telomeres via TIN2, where it functioned as a negative regulator of telomerase-mediated telomere elongation. TIN2 associated with TINT1, and TRF1 or TRF2 throughout the cell cycle, revealing a partially redundant unit in telomeric chromatin that may provide flexibility in telomere length control. Indeed, when TRF1 was removed from telomeres by overexpression of the positive telomere length regulator tankyrase 1, the TIN2/TINT1 complex remained on telomeres via an increased association with TRF2. CONCLUSIONS: Our findings suggest a dynamic cross talk between TRF1 and TRF2 and provide a molecular mechanism for telomere length homeostasis by TRF2 in the absence of TRF1

Simple (i.e., nonstratified) epithelial cells use two different routes to target their newly synthesized luminal plasma membrane proteins to the cell surface: a direct route from the Golgi complex, as in the kidney-derived MDCK cell line, or an indirect route that involves a intermediate stop at the ab-luminal (basolateral) membrane, as in hepatocytes. The mechanisms or proteins responsible for these different protein targeting strategies are not known. Here, we show that increased expression of EMK1, a mammalian ortholog of Caenorhabditis elegans Par-1, in MDCK cells promotes a switch from a direct to a transcytotic mode of apical protein delivery and other trafficking changes typical of hepatocytes. These results, together with our recent demonstration that PAR-1 promotes morphological features of hepatocytes in MDCK cells, indicate that Par-1 modulates the developmental decision to build a columnar versus a hepatic epithelial cell. To our knowledge, Par-1 is the first gene assigned to this task in epithelial morphogenesis.

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs

Genomes hold within them the record of the evolution of life on Earth. But genome fusions and horizontal gene transfer seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life. Here we determine the general outline of the tree using complete genome data from representative prokaryotes and eukaryotes and a new genome analysis method that makes it possible to reconstruct ancient genome fusions and phylogenetic trees. Our analyses indicate that the eukaryotic genome resulted from a fusion of two diverse prokaryotic genomes, and therefore at the deepest levels linking prokaryotes and eukaryotes, the tree of life is actually a ring of life. One fusion partner branches from deep within an ancient photosynthetic clade, and the other is related to the archaeal prokaryotes. The eubacterial organism is either a proteobacterium, or a member of a larger photosynthetic clade that includes the Cyanobacteria and the Proteobacteria.

The visualization of beta-amyloid plaque deposition in brain, a key feature of Alzheimer's disease (AD), is important for the evaluation of disease progression and the efficacy of therapeutic interventions. In this study, beta-amyloid plaques in the PS/APP transgenic mouse brain, a model of human AD pathology, were detected using MR microscopy without contrast reagents. beta-Amyloid plaques were clearly visible in the cortex, thalamus, and hippocampus of fixed brains of PS/APP mice. The distribution of plaques identified by MRI was in excellent agreement with those found in the immunohistological analysis of the same brain sections. It was also demonstrated that image contrast for beta-amyloid plaques was present in freshly excised nonfixed brains. Furthermore, the detection of beta-amyloid plaques was achieved with a scan time as short as 2 hr, approaching the scan time considered reasonable for in vivo imaging

An association exists among neurofibromatosis 1 (NF1), juvenile xanthogranulomas (JXGs), and juvenile myelomonocytic leukemia (JMML). The authors describe a patient with the triple association of JXG, NF1, and JMML initially presenting with features of hemophagocytic lymphohistiocytosis (HLH). An 18-month old boy had multiple cutaneous and gastrointestinal JXG and NF1. At 3 years of age he developed anemia, thrombocytopenia, and hepatosplenomegaly. A bone marrow biopsy revealed features of HLH. Despite chemotherapy, he went on to develop JMML, which proved fatal

Plasmodium liver stages or early exo-eythrocytic forms (EEFs) contain antigens that are essential for achieving sterile, protective immunity against malaria. Yet, attempts at identifying these antigens have been hampered by the challenge of obtaining large numbers of purified EEFs, uncontaminated with hepatocyte material. Using a recently described system for producing axenically cultured EEFs from Plasmodium yoelii, we have constructed a cDNA library and generated 1453 expressed sequence tags (ESTs) resulting in 652 unique transcripts. Analysis of the library provides insight into processes required for the initiation and development of Plasmodium liver stages, such as protein degradation, cell cycle progression and nutrient transport. Analysis of the gene expression profile of liver stages, as revealed by this library, suggests that liver stages represent a shift from 'sporozoite-like' to 'blood-stage-like'. This is the first study of the transcriptional repertoire of Plasmodium liver stages

The activity of both human tissue-type plasminogen activator (t-PA) and the PA from the saliva of the vampire bat, Desmodus rotundus, (DSPA) is critically dependent on the presence of a cofactor. The most efficient cofactor for both PAs is fibrin, but fibrinogen and amyloid beta peptides also have cofactor activities for human t-PA. Compared to t-PA, DSPA has a more stringent requirement for fibrin as a cofactor. The present study was undertaken to compare cofactor activities of amyloid beta 1-42 (Abeta1-42) for plasminogen activation by DSPA-alpha1 or by t-PA. The two PAs were incubated with different concentrations of glu-plasminogen, a chromogenic substrate for plasmin and 100 micro g mL (-1) of Abeta1-42, fibrinogen or fibrin as cofactor. Using the kinetic parameters directly determined from the chromogenic substrate conversion curves, we derived the relative efficacies of DSPA or t-PA in the presence of cofactor at the physiological plasminogen concentration of 2 micro M. In the presence of fibrin, the activity of DSPA was comparable to that of t-PA and 23,270-fold higher than its activity without cofactor, whereas fibrin induced only a 248-fold increase in t-PA activity. The activity of DSPA with Abeta1-42 or fibrinogen as cofactor was 485-fold lower than its activity in the presence of fibrin, while for t-PA this difference was only 26-fold. The much lower activity of DSPA as compared to t-PA with Abeta1-42 or fibrinogen might lead to fewer side effects when used for the thrombolytic therapy of stroke.

OBJECTIVE: We determined the effects of sustained normocholesterolemia on advanced mouse atherosclerosis and whether changes in plaque size and composition can be detected noninvasively by MRI. METHODS AND RESULTS: Aortic arch segments containing advanced lesions from apolipoprotein E-deficient (apoE-/-) mice (total cholesterol 1281+/-97 mg/dL) were transplanted into syngeneic wild-type (WT; 111+/-11 mg/dL) or apoE-/- (702+/-74 mg/dL) recipient mice on chow diet. Mice underwent serial MRI at 3, 5, 7, and 9 weeks after transplantation. Compared with 3 weeks, correction of dyslipidemia in WT recipient mice resulted in a monotonic decrease (regression) in arterial wall volume, whereas in apoE-/- recipient mice, further plaque progression was noted (P<0.05). MRI and histological measurements were closely correlated (R=0.937). The lesional content of macrophages decreased >90% (P<0.001), and smooth muscle cells increased in the WT recipient mice. In vivo T(1)-, T(2)-, and proton density-weighted images of the mouse thoracic aorta differentiated intraplaque lipid and collagen. CONCLUSIONS: Plaque changes can be noninvasively monitored by serial in vivo MRI of a mouse regression model. Our ability to image the thoracic aorta and perform in vivo plaque characterization will further enhance atherosclerosis studies. Serial in vivo MRI of mouse arterial plaque after correction of dyslipidemia revealed a monotonic decrease in lesion size (regression) and changes in lesion composition consistent with a stable plaque phenotype. Serial in vivo MRI will enhance studies of plaque regression in animal models in response to therapeutic interventions