Coculturing diverse clonal populations prevents the early-stage neoplastic progression that occurs in the separate clones

Most human cancers are of monoclonal origin and display many genetic alterations. In an effort to determine whether clonal expansion itself could account for the large number of genetic alterations, we compared spontaneous transformation in cloned and uncloned populations of NIH 3T3 cells. We have reported that progressive transformation of these cells, which is driven by the stress of prolonged contact inhibition at confluence, occurs far more frequently in cultures of recent monoclonal origin than in their uncloned progenitors. In the present work we asked how coculturing six clones at early and late stages of progression would affect the dynamics of transformation in repeated rounds of confluence. When coculture started with clones in early stages of transformation, marked by light focus formation, there was a strong inhibition of the progression to the dense focus formation that occurred in separate cultures of the individual clones. In contrast, when coculture started after the individual clones had progressed to dense focus formation, there was selection of transformants from the clone producing the largest and densest foci. Mixing the cells of a single clone with a large excess of uncloned cells from a subline that was refractory to transformation markedly decreased the size of dense foci from clones in transit from light to dense focus formation, but had much less effect on foci from clones with an established capacity for dense focus formation. The major finding of protection against progression by coculturing clones in early stages of transformation suggests that the expansion of a rogue clone in vivo increasingly isolates many of its cells from genetically stabilizing interactions with surrounding clones. Such clonal isolation might account for the increase in mutation rates associated with the dysplasia in colorectal adenomas that signifies the transition between benign and malignant growth.

Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2

The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase, Shp2 homozygous mutant embryonic stem cells bearing the Rosa-26 LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that Shp2 mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage, Shp2 mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of Shp2 mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor in vitro. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4−CD8− double negative (DN) cells to CD4+CD8+ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

Folding and aggregation of designed proteins

Protein aggregation is studied by following the simultaneous folding of two designed identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that protein aggregation is determined by elementary structures (partially folded intermediates) controlled by local contacts among some of the most strongly interacting amino acids and formed at an early stage in the folding process.

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

Amyloid-β peptide–Receptor for Advanced Glycation Endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: A proinflammatory pathway in Alzheimer disease

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Aβ) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Aβ, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor κB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Aβ deposits, and in cerebrospinal fluid from AD patients there was ≈5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Aβ-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Aβ, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Aβ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network

We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied in vitro using vesicular stomatitis virus-infected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN.

Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Δ cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF–RARα and NPM–RARα

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARα and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF–RARα and NPM–RARα. PLZF–RARα transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM–RARα transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF–RARα transgenic mice, those from NPM–RARα transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 μM, 0.1 μM, and 1.0 μM for RARα–RXRα, NPM–RARα, and PML–RARα, respectively, but not observed for PLZF–RARα even in the presence of 10 μM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF–RARα and NPM–RARα and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.

Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins

Aβ1–42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.

Regulation of CD40 function by its isoforms generated through alternative splicing

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-κB, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

A minimal gene set for cellular life derived by comparison of complete bacterial genomes.

The recently sequenced genome of the parasitic bacterium Mycoplasma genitalium contains only 468 identified protein-coding genes that have been dubbed a minimal gene complement [Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., et al. (1995) Science 270, 397-403]. Although the M. genitalium gene complement is indeed the smallest among known cellular life forms, there is no evidence that it is the minimal self-sufficient gene set. To derive such a set, we compared the 468 predicted M. genitalium protein sequences with the 1703 protein sequences encoded by the other completely sequenced small bacterial genome, that of Haemophilus influenzae. M. genitalium and H. influenzae belong to two ancient bacterial lineages, i.e., Gram-positive and Gram-negative bacteria, respectively. Therefore, the genes that are conserved in these two bacteria are almost certainly essential for cellular function. It is this category of genes that is most likely to approximate the minimal gene set. We found that 240 M. genitalium genes have orthologs among the genes of H. influenzae. This collection of genes falls short of comprising the minimal set as some enzymes responsible for intermediate steps in essential pathways are missing. The apparent reason for this is the phenomenon that we call nonorthologous gene displacement when the same function is fulfilled by nonorthologous proteins in two organisms. We identified 22 nonorthologous displacements and supplemented the set of orthologs with the respective M. genitalium genes. After examining the resulting list of 262 genes for possible functional redundancy and for the presence of apparently parasite-specific genes, 6 genes were removed. We suggest that the remaining 256 genes are close to the minimal gene set that is necessary and sufficient to sustain the existence of a modern-type cell. Most of the proteins encoded by the genes from the minimal set have eukaryotic or archaeal homologs but seven key proteins of DNA replication do not. We speculate that the last common ancestor of the three primary kingdoms had an RNA genome. Possibilities are explored to further reduce the minimal set to model a primitive cell that might have existed at a very early stage of life evolution.