A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

We have tested two of the hypotheses proposed to explain the adjustment in sodium reabsorption in the proximal tubule that follows a change in the rate of glomerular filtration (glomerulotubular balance). Using the recollection micropuncture technique, we were able to measure the immediate and late changes in reabsorptive rate after an acute alteration in filtration rate produced by aortic constriction and release of constriction. It was found that fractional reabsorption, as measured by the inulin tubule fluid to plasma (TF/P) ratio, increased after aortic constriction and decreased after release, but that in most instances, absolute reabsorptive rate changed in parallel to glomerular filtration rate. The change was similar whether the collections were made less than 1 or more than 5 min after the change in blood pressure. The rapid time course of this adjustment in reabsorptive rate is viewed as evidence against an intrarenal humoral feedback mechanism. In the same experiments we measured the (TF/P)(In), transit time, and flow rate of fluid in single nephrons before and during aortic constriction or release of aortic constriction. The change in reabsorptive rate and the simultaneous change in calculated cross-sectional area of the tubule lumen were rarely proportional, i.e., C/pir(2) was not constant. In other experiments, these same measurements were made before and during periods of increased ureteral pressure. Despite large increments in calculated cross-sectional area, the absolute rate of reabsorption either remained relatively unchanged or fell in proportion to the change in filtration rate. It is concluded that under these conditions, reabsorptive rate is governed by some factor other than tubule geometry.

Thrombin, generated through activation of factor XI (FXI) and/or tissue factor (TF)-factor VIIa, is essential for thrombosis and hemostasis. We investigated the role of FXI-dependent thrombus propagation under arterial flow conditions producing rapid thrombus growth that, after the initiation phase, could limit the availability of TF at the blood/thrombus interface. Thrombosis was initiated by knitted dacron or TF-presenting teflon grafts deployed into arteriovenous shunts in baboons treated with antihuman FXI antibody (aFXI). Although aFXI did not prevent thrombus initiation, it markedly reduced intraluminal thrombus growth on both surfaces. The antithrombotic effect of aFXI was comparable with that of heparin at doses that significantly prolonged the partial thromboplastin time (APTT), prothrombin time (PT), and bleeding time (BT). aFXI also prolonged the APTT, but the PT and BT were unaffected. Thus, antithrombotic targeting of FXI might inhibit thrombosis with relatively modest hemostatic impairment versus strategies targeting other coagulation factors.

HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR), the receptor used by cells to obtain iron in the form of diferric transferrin (Fe-Tf). Previous studies demonstrated that HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, and that membrane-bound or soluble HFE binding to cell surface TfR results in a reduction in the affinity of TfR for Fe-Tf. We studied the inhibition by soluble HFE of the interaction between soluble TfR and Fe-Tf using radioactivity-based and biosensor-based assays. The results demonstrate that HFE inhibits the TfR:Fe-Tf interaction by binding at or near the Fe-Tf binding site on TfR, and that the Fe-Tf:TfR:HFE ternary complex consists of one Fe-Tf and one HFE bound to a TfR homodimer.

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

A majority of tissue factor (TF) on cell surfaces exists in a cryptic form (ie, coagulation function inactive) but retains its functionality in cell signaling. Recent studies have suggested that cryptic TF contains unpaired cysteine thiols and that activation involves the formation of the disulfide bond Cys186-Cys 209 and that protein disulfide isomerase (PDI) regulates TF coagulant and signaling activities by targeting this disulfide bond. This study was carried out to investigate the validity of this novel concept. Although treatment of MDA 231 tumor cells, fibroblasts, and stimulated endothelial cells with the oxidizing agent HgCl(2) markedly increased the cell-surface TF coagulant activity, the increase is associated with increased anionic phospholipids at the cell surface. Annexin V, which binds to anionic phospholipids, attenuated the increased TF coagulant activity. It is noteworthy that treatment of cells with reducing agents also increased the cell surface TF activity. No evidence was found for either detectable expression of PDI at the cell surface or association of TF with PDI. Furthermore, reduction of PDI with the gene silencing had no effect on either TF coagulant or cell signaling functions. Overall, the present data undermine the recently proposed hypothesis that PDI-mediated disulfide exchange plays a role in regulating TF procoagulant and cell signaling functions.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.

Behaviors are often highly heritable, polygenic traits. To investigate molecular mediators of behavior, we analyzed gene expression patterns across seven brain regions (amygdala, basal ganglia, cerebellum, frontal cortex, hippocampus, cingulate cortex, and olfactory bulb) of 10 different inbred mouse strains (129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and FVB/NJ). Extensive variation was observed across both strain and brain region. These data provide potential transcriptional intermediates linking polygenic variation to differences in behavior. For example, mice from different strains had variable performance on the rotarod task, which correlated with the expression of >2000 transcripts in the cerebellum. Correlation with this task was also found in the amygdala and hippocampus, but not in other regions examined, indicating the potential complexity of motor coordination. Thus we can begin to identify expression profiles contributing to behavioral phenotypes through variation in gene expression.

The BTBR T+ tf/J (BTBR) inbred mouse strain displays a low sociability phenotype relevant to the first diagnostic symptom of autism, deficits in reciprocal social interactions. Previous studies have shown that BTBR mice exhibit reduced social approach, juvenile play, and interactive behaviors. The present study evaluated the behavior of the BTBR and C57BL/6J (B6) strains in social proximity. Subjects were closely confined and tested in four experimental conditions: same strain male pairs (Experiment 1); different strain male pairs (Experiment 2); same strain male pairs and female pairs (Experiment 3); same strain male pairs treated with an anxiolytic (Experiment 4). Results showed that BTBR mice displayed decreased nose tip-to-nose tip, nose-to-head and upright behaviors and increased nose-to-anogenital, crawl under and crawl over behaviors. These results demonstrated avoidance of reciprocal frontal orientations in the BTBR, providing a parallel to gaze aversion, a fundamental predictor of autism. For comparative purposes, Experiment 3 assessed male and female mice in a three-chamber social approach test and in the social proximity test. Results from the three-chamber test showed that male B6 and female BTBR displayed a preference for the sex and strain matched conspecific stimulus, while female B6 and male BTBR did not. Although there was no significant interaction between sex and strain in the social proximity test, a significant main effect of sex indicated that female mice displayed higher levels of nose tip-to-nose tip contacts and lower levels of anogenital investigation (nose-to-anogenital) in comparison to male mice, all together suggesting different motivations for sociability in males and females. Systemic administration of the anxiolytic, diazepam, decreased the frequency of two behaviors associated with anxiety and defensiveness, upright and jump escape, as well as crawl under behavior. This result suggests that crawl under behavior, observed at high levels in BTBR mice, is elicited by the aversiveness of social proximity, and possibly serves to avoid reciprocal frontal orientations with other mice.

The ability of cells to accurately control gene expression levels in response to extracellular cues is limited by the inherently stochastic nature of transcriptional regulation. A change in transcription factor (TF) activity results in changes in the expression of its targets, but the way in which cell-to-cell variability in expression (noise) changes as a function of TF activity, and whether targets of the same TF behave similarly, is not known. Here, we measure expression and noise as a function of TF activity for 16 native targets of the transcription factor Zap1 that are regulated by it through diverse mechanisms. For most activated and repressed Zap1 targets, noise decreases as expression increases. Kinetic modeling suggests that this is due to two distinct Zap1-mediated mechanisms that both change the frequency of transcriptional bursts. Notably, we found that another mechanism of repression by Zap1, which is encoded in the promoter DNA, likely decreases the size of transcriptional bursts, producing a unique transcriptional state characterized by low expression and low noise. In addition, we find that further reduction in noise is achieved when a single TF both activates and represses a single target gene. Our results suggest a global principle whereby at low TF concentrations, the dominant source of differences in expression between promoters stems from differences in burst frequency, whereas at high TF concentrations differences in burst size dominate. Taken together, we show that the precise amount by which noise changes with expression is specific to the regulatory mechanism of transcription and translation that acts at each gene.

Hahb-4 is a member of the Helianthusannuus (sunflower) subfamily I of HD-Zip proteins that is transcriptionally regulated by water availability and abscisic acid. Transgenic Arabidopsis thaliana plants overexpressing this transcription factor (TF) exhibit a characteristic phenotype that includes a strong tolerance to water stress. Here we show that this TF is a new component of ethylene signalling pathways, and that it induces a marked delay in senescence. Plants overexpressing Hahb-4 are less sensitive to external ethylene, enter the senescence pathway later and do not show the typical triple response. Furthermore, transgenic plants expressing this gene under the control of its own inducible promoter showed an inverse correlation between ethylene sensitivity and Hahb-4 levels. Potential targets of Hahb-4 were identified by comparing the transcriptome of Hahb-4-transformed and wild-type plants using microarrays and quantitative RT-PCR. Expression of this TF has a major repressive effect on genes related to ethylene synthesis, such as ACO and SAM, and on genes related to ethylene signalling, such as ERF2 and ERF5. Expression studies in sunflower indicate that Hahb-4 transcript levels are elevated in mature/senescent leaves. Expression of Hahb-4 is induced by ethylene, concomitantly with several genes homologous to the targets identified in the transcriptome analysis (HA-ACOa and HA-ACOb). Transient transformation of sunflower leaves demonstrated the action of Hahb-4 in the regulation of ethylene-related genes. We propose that Hahb-4 is involved in a novel conserved mechanism related to ethylene-mediated senescence that functions to improve desiccation tolerance.

The transmembrane cellular receptor tissue factor (TF) is the primary initiator of the coagulation cascade in man. Expression of TF was determined in 55 specimens of ductal adenocarcinoma of the pancreas and was found to correlate strongly with the degree of histological differentiation. A significant linear trend was observed with stronger immunoreactivity observed in poorly differentiated tumours (chi 2 = 6.69, P = 0.0098). No TF staining was seen in pancreatic samples from normal controls (n = 18). As expression of TF may be associated with tumour progression, its analysis could provide useful prognostic information in patients with pancreatic malignancy.

Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

Inbred mouse strains differ greatly in social behaviors, making them a valuable resource to study genetic and non-genetic mechanisms underlying social deficits relevant to autism spectrum disorders. A hallmark symptom of autism is a lack of ability to understand other people's thoughts and intentions, which leads to impairments in adjusting behaviors in response to ever-changing social situations in daily life. We compared the ability of BTBR T+tf/J (BTBR), a strain with low sociability, and C57BL/6J (B6), a strain with high sociability, for their abilities to modulate responses to social cues from different partners in the reciprocal social interaction test. Results indicate that BTBR exhibited low sociability toward different partners and displayed minimal ability to modify behaviors toward different partners. In contrast, B6 showed high sociability toward different partners and was able to modify social behaviors toward different partners. Consistent results were found in two independent cohorts of different ages, and in both sexes. In the three-chambered test, high sociability in B6 and low sociability in BTBR were independent of strain of the novel mouse. Since social deficits in BTBR could potentially be caused by physical disabilities in detecting social olfactory cues, or in cognitive abilities, we tested BTBR and B6 mice on measures of olfaction and cognition. BTBR mice displayed more sniffing of social odors emitted by soiled bedding than of an odorless novel object, but failed to show a preference for a live novel mouse over a novel object. On olfactory habituation/dishabituation to a sequence of odors, BTBR displayed discrimination abilities across three non-social and two social odors. However, as compared to B6, BTBR displayed less sniff time for both non-social and social odors, and no significant dishabituation between cage odors from two different novel mouse strains, findings that will be important to investigate further. BTBR was generally normal in spatial acquisition on the Morris water maze test, but showed deficits in reversal learning. Time spent freezing on contextual and cued fear conditioning was lower in BTBR than in B6. Our findings suggest that BTBR has poor abilities to modulate its responses to different social partners, which may be analogous to social cognition deficits in autism, adding to the value of this strain as a mouse model of autism.

The use of cationic liposomes as nonviral vehicles for the delivery of therapeutic molecules is becoming increasingly prevalent in the field of gene therapy. We have previously demonstrated that the use of the transferrin ligand (Tf) to target a cationic liposome delivery system resulted in a significant increase in the transfection efficiency of the complex [Xu, L., Pirollo, K.F., and Chang, E.H. (1997). Hum. Gene Ther. 8, 467-475]. Delivery of wild-type (wt) p53 to a radiation-resistant squamous cell carcinoma of the head and neck (SCCHN) cell line via this ligand-targeted, liposome complex was also able to revert the radiation resistant phenotype of these cells in vitro. Here we optimized the Tf/liposome/DNA ratio of the complex (LipT) for maximum tumor cell targeting, even in the presence of serum. The efficient reestablishment of wtp53 function in these SCCHN tumor cells in vitro, via the LipT complex, restored the apoptotic pathway, resulting in a significant increase in radiation-induced apoptosis that was directly proportional to the level of exogenous wtp53 in the tumor cells. More significantly, intravenous administration of LipT-p53 markedly sensitized established SCCHN nude mouse xenograft tumors to radiotherapy. The combination of systemic LipT-p53 gene therapy and radiation resulted in complete tumor regression and inhibition of their recurrence even 6 months after the end of all treatment. These results indicate that this tumor-specific, ligand-liposome delivery system for p53 gene therapy, when used in concert with conventional radiotherapy, can provide a new and more effective means of cancer treatment.

Tissue factor (TF)-initiated coagulation plays a significant role in the pathophysiology of many diseases, including cancer and inflammation. Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor, which modulates initiations of coagulation induced by TF. In a factor (F) Xa-dependent feedback system, TFPI binds directly and inhibits the TF-FVII/FVIIa complex. Normally, TFPI exists in plasma both as a full-length molecule and as variably carboxy-terminal truncated forms. TFPI also circulates in complex with plasma lipoproteins. The levels and the dual inhibitor effect of TFPI on FXa and TF-FVII/FVIIa complex offers insight into the mechanisms of various pathological conditions triggered by TF. The use of selective pharmacological inhibitors has become an indispensable tool in experimental haemostasis and thrombosis research. In vivo administration of recombinant TFPI (rTFPI) in an experimental animal model prevents thrombosis (and re-thrombosis after thrombolysis), reduces mortality from E. coli-induced-septic shock, prevents fibrin deposition on subendothelial human matrix and protects against disseminated intravascular coagulation (DIC). Thus, TFPI may play an important role in modulating TF-induced thrombogenesis and it may also provide a unique therapeutic approach for prophylaxis and/or treatment of various diseases. In this review, we consider structural and biochemical aspects of the TFPI molecule and detail its inhibitory mechanisms and therapeutic implications in various disease conditions.

Chlamydia trachomatis, an obligate intracellular parasite and a major human pathogen, invades eukaryotic host cells and replicates within a membrane-bound compartment (termed the vacuole or inclusion) in the cytoplasm of the host cell. In this report, we describe in detail the characteristics of the vacuole throughout the chlamydial life cycle in terms of the endocytic pathway, as determined by epifluorescent and confocal immunofluorescence microscopy. By indirect immunofluorescence, the transferrin receptor (TfR), a component of early endosomes, and the cation-independent mannose-6-phosphate receptor (CI-M6PR), a component of late endosomes, were found in close association with the chlamydial vacuole as early as 4 h postinfection (hpi) and as late as 20 hpi. Fluorescein isothiocyanate (FITC)-labeled Tf was also found to colocalize with the vacuole at 4, 12, and 20 hpi, indicating that exogenously added ligands can be transported to the region of the vacuole. Antibodies to several different lysosomal proteins failed to label the chlamydial vacuole at any time point during the life cycle. Indirect immunofluorescence of cells infected with chlamydiae stained with an antibody to the trans-Golgi network (TGN) protein TGN38 demonstrated that in infected cells, the integrity and structure of the TGN was altered. The rates of Tf recycling in infected and uninfected cells were compared by fluorescence microscopy and quantitated with 125I-Tf. While the rate of FITC-Tf recycling from endocytic compartments in chlamydia-infected cells did not appear different from that of uninfected cells, a small pool of FITC-Tf that had accumulated adjacent to the chlamydial vacuole recycled at a slower rate. Quantitation of Tf recycling with 125I-Tf showed that Tf was recycled more slowly in infected cells than in uninfected cells. The altered distribution of several endocytic pathway markers and the slowed Tf recycling are consistent with the hypothesis that the chlamydial vacuole interacts with the endocytic pathway of the host. These results furthermore suggest that the chlamydial vacuole does not correspond to a canonical endocytic compartment but that it is a unique and dynamic organelle that shares several characteristics with recycling endosomes of the host cell. Interactions with the early and/or late endosomal compartments, in addition to the Golgi apparatus, may provide a source of membrane or nutrients for the replicating organisms.

Developmental processes require a precise spatio-temporal regulation of gene expression wherein a diverse set of transcription factors control the signalling pathways. MicroRNAs (miRNAs), a class of small non-coding RNA molecules have recently drawn attention for their prominent role in development and disease. These tiny sequences are essential for regulation of processes, including cell signalling, cell development, cell death, cell proliferation, patterning and differentiation. The consequence of gene regulation by miRNAs is similar to that by transcription factors (TFs). A regulatory cascade essential for appropriate execution of several biological events is triggered through a combinatorial action of miRNAs and TFs. These two important regulators share similar regulatory logics and bring about a cooperative action in the gene regulatory network, dependent on the binding sites present on the target gene. The review addresses the biogenesis and nomenclature of miRNAs, outlines the mechanism of action and regulation of their expression, and focuses on the combinatorial action of miRNAs and TFs for the expression of genes in various regulatory cascades.

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

OBJECTIVE

To ascertain whether the differential responses that previously have been found between trauma-focused, cognitive-behavioral therapy (TF-CBT), and child-centered therapy (CCT) for treating posttraumatic stress disorder (PTSD) and related problems in children who had been sexually abused would persist following treatment and to examine potential predictors of treatment outcome.

METHODS

A total of 183 children 8 to 14 years old and their primary caregivers were assessed 6 and 12 months after their posttreatment evaluations.

RESULTS

Mixed-model repeated analyses of covariance found that children treated with TF-CBT had significantly fewer symptoms of PTSD and described less shame than the children who had been treated with CCT at both 6 and 12 months. The caregivers who had been treated with TF-CBT also continued to report less severe abuse-specific distress during the follow-up period than those who had been treated with CCT. Multiple traumas and higher levels of depression at pretreatment were positively related to the total number of PTSD symptoms at posttreatment for children assigned to the CCT condition only.

CONCLUSIONS

Children and caregivers assigned to TF-CBT continued to have fewer symptoms of PTSD, feelings of shame, and abuse-specific parental distress at 6- and 12-month assessments as compared to participants assigned to CCT.

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

Yeast strains with mutations in both TEL1 and MEC1 have short telomeres and elevated rates of chromosome deletions. By using a PCR assay, we demonstrate that mec1 tel1 strains also have telomere-telomere fusions (T-TFs). T-TFs require Lig4p (a ligase required for nonhomologous end-joining DNA repair). The highest rates of T-TFs are found in strains with combination of mutations that affect telomere length and DNA damage checkpoints (mec1 tel1, mec3 tel1, mre11 mec1, and ddc1 tel1 strains). Examining many mutant genotypes, we find good agreement between the level of T-TFs and the rate of chromosomal deletions. In addition, if telomeres are elongated in a mec1 tel1 strain, we eliminate T-TFs and reduce the deletion rate. The correlation between the level of T-TFs and the rate of deletions argues that many of these deletions reflect a cycle of T-TF formation (resulting in dicentric chromosomes), followed by chromosome breakage.

BACKGROUND

Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations.

METHODS

OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments.

RESULTS

OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew.

CONCLUSIONS

Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.