Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.

The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergent-insoluble microdomains that inefficiently support TF-VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.

Abiotic stress causes loss of crop production. Under abiotic stress conditions, expression of many genes is induced, and their products have important roles in stress responses and tolerance. Progress has been made in understanding the biological roles of regulons in abiotic stress responses in rice. A number of transcription factors (TFs) regulate stress-responsive gene expression. OsDREB1s and OsDREB2s were identified as abiotic-stress responsive TFs that belong to the AP2/ERF family. Similar to Arabidopsis, these DREB regulons were most likely not involved in the abscisic acid (ABA) pathway. OsAREBs such as OsAREB1 were identified as key components in ABA-dependent transcriptional networks in rice. OsNAC/SNACs including OsNAC6 were characterized as factors that regulate expression of genes important for abiotic stress responses in rice. Here, we review on the rice abiotic-stress responses mediated by transcriptional networks, with the main focus on TFs that function in abiotic stress responses and confer stress tolerance in rice.

OBJECTIVE

Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multicenter analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as an overall (OS) and treatment-free survival (TFS) predictor.

METHODS

A training/validation strategy was chosen to find the optimal CD49d cutoff. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLLs; Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (eg, CD38, ZAP-70) was ranked by recursive partitioning.

RESULTS

Patients with ≥ 30% of neoplastic cells expressing CD49d were considered CD49d+. Decrease in OS at 5 and 10 years among CD49d+ patients was 7% and 23% (decrease in TFS, 26% and 25%, respectively). Pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR, 2.0) in a Cox model adjusted for clinical and biologic prognosticators. Hierarchic trees including all patients or restricted to those with early-stage disease or those age ≤ 65 years always selected CD49d as the most important flow cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patient subsets with poorer outcome independent of CD38 and ZAP-70.

CONCLUSIONS

In this analysis of approximately 3,000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL.

The effective treatment of malignant brain glioma is hindered by the poor transport across the blood-brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). In this study, transferrin-conjugated magnetic silica PLGA nanoparticles (MNP-MSN-PLGA-Tf NPs) were formulated to overcome these barriers. These NPs were loaded with doxorubicin (DOX) and paclitaxel (PTX), and their anti-proliferative effect was evaluated in vitro and in vivo. The in vitro cytotoxicity of drug-loaded NPs was evaluated in U-87 cells. The delivery and the subsequent cellular uptake of drug-loaded NPs could be enhanced by the presence of magnetic field and the usage of Tf as targeting ligand, respectively. In particular, cells treated with DOX-PTX-NPs-Tf with magnetic field showed the highest cytotoxicity as compared to those treated with DOX-PTX-NPs-Tf, DOX-PTX-NPs, DOX-PTX-NPs-Tf with free Tf. The in vivo therapeutic efficacy of drug-loaded NPs was evaluated in intracranial U-87 MG-luc2 xenograft of BALB/c nude mice. In particular, the DOX-PTX-NPs-Tf treatment exhibited the strongest anti-glioma activity as compared to the PTX-NPs-Tf, DOX-NPs-Tf or DOX-PTX-NPs treatment. Mice did not show acute toxicity after administrating with blank MNP-MSN-PLGA-Tf NPs. Overall, MNP-MSN-PLGA-Tf NPs are promising carriers for the delivery of dual drugs for effective treatment of brain glioma.

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415-5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1alpha, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

BACKGROUND

Several studies suggest a role for an increased circulating pool of tissue factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating tissue factor activity. We have extended our study to smokers and hyperlipidemic subjects.

RESULTS

Poorly controlled patients with type 2 diabetes mellitus (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by factor Xa (FXa) generation in the presence of factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber. Patients with improvement in glycemic control showed a reduction in circulating TF (362+/-135 versus 243+/-74 pmol/L per min FXa, P=0.0001). A similar effect was observed in BT (15 445+/-1130 versus 12 072+/-596 microm/mm2, P=0.01). Two hours after smoking 2 cigarettes, TF was increased (217+/-72 versus 283+/-106 pmol/L per min FXa, P=0.003). Hyperlipidemic subjects showed higher TF (237+/-63 versus 195+/-44 pmol/L per min FXa, P=0.035) than healthy volunteers.

CONCLUSIONS

These findings suggest that high levels of circulating TF may be the mechanism of action responsible for the increased thrombotic complications associated with the presence of these cardiovascular risk factors. These observations strongly emphasize the usefulness of the management of the patients based on their global risk assessment.

BACKGROUND

Transcription factors (TFs) are crucial during the lifetime of the cell. Their functional roles are defined by the genes they regulate. Uncovering these roles not only sheds light on the TF at hand but puts it into the context of the complete regulatory network.

RESULTS

Here, we present an alignment- and threshold-free comparative genomics approach for assigning functional roles to DNA regulatory motifs. We incorporate our approach into the Gomo algorithm, a computational tool for detecting associations between a user-specified DNA regulatory motif [expressed as a position weight matrix (PWM)] and Gene Ontology (GO) terms. Incorporating multiple species into the analysis significantly improves Gomo's ability to identify GO terms associated with the regulatory targets of TFs. Including three comparative species in the process of predicting TF roles in Saccharomyces cerevisiae and Homo sapiens increases the number of significant predictions by 75 and 200%, respectively. The predicted GO terms are also more specific, yielding deeper biological insight into the role of the TF. Adjusting motif (binding) affinity scores for individual sequence composition proves to be essential for avoiding false positive associations. We describe a novel DNA sequence-scoring algorithm that compensates a thermodynamic measure of DNA-binding affinity for individual sequence base composition. GOMO's prediction accuracy proves to be relatively insensitive to how promoters are defined. Because GOMO uses a threshold-free form of gene set analysis, there are no free parameters to tune. Biologists can investigate the potential roles of DNA regulatory motifs of interest using GOMO via the web (http://meme.nbcr.net).

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.

Lupus nephritis (LN) is among the main determinants of poor prognosis in systemic lupus erythematosus (SLE). The objective of this study was to 1) isolate and identify proteins contained in the LN urinary protein signature (PS) of children with SLE; 2) assess the usefulness of the PS proteins for detecting activity of LN over time. Using surface-enhanced or matrix-assisted laser desorption/ionization time of flight mass spectrometry, the proteins contained in the LN urinary PS were identified. They were transferrin (Tf), ceruloplasmin (Cp), alpha1-acid-glycoprotein (AGP), lipocalin-type prostaglandin-D synthetase (L-PGDS), albumin, and albumin-related fragments. Serial plasma and urine samples were analyzed using immunonephelometry or ELISA in 98 children with SLE (78% African American) and 30 controls with juvenile idiopathic arthritis. All urinary PS proteins were significantly higher with active vs. inactive LN or in patients without LN (all p < 0.005), and their combined area under the receiver operating characteristic curve was 0.85. As early as 3 mo before a clinical diagnosis of worsening LN, significant increases of urinary Tf, AGP (both p < 0.0001), and L-PGDS (p < 0.01) occurred, indicating that these PS proteins are biomarkers of LN activity and may help anticipate the future course of LN.

To elucidate the pathophysiology of idiopathic pulmonary fibrosis (IPF), we examined procoagulant (tissue factor:TF), fibrinolytic (tissue type plasminogen activator:t-PA and urokinase type plasminogen activator:u-PA) and antifibrinolytic (plasminogen activator inhibitor-1:PAI-1 and PAI-2) activities in bronchoalveolar lavage (BAL) supernatant fluids and BAL cell lysates obtained from IPF patients. The results indicated that TF levels in BAL supernatant fluids from IPF patients were higher than those of normal subjects, especially in patients with progressive disease, suggesting that TF levels in the lung correlate with disease activity. PAI-1 levels in BAL supernatant fluids were significantly higher in IPF patients than in normal subjects (1.7 +/- 4.1 vs 0 ng/mg protein). PAI-2 levels in BAL cell lysates were also significantly higher in IPF patients than those in normal subjects (14.4 +/- 12.2 vs 3.0 +/- 3.0 ng/mg protein). However, u-PA levels in both BAL supernatant fluids and BAL cell lysates did not differ between the two groups. These observations suggest that u-PA inhibition exceeded u-PA activity in alveolar lining fluid resulting in an antifibrinolytic condition. Immunohistochemical analysis showed that TF was intensely stained in cuboidal epithelial cells and PAIs were positively stained in alveolar macrophages (AMs) and cuboidal epithelial cells, suggesting that cuboidal epithelial cells as well as AMs contribute to the increased procoagulant and antifibrinolytic activities in the lungs of IPF patients.

OBJECTIVE

Ultrasonography allows the direct observation of the diaphragm. Its thickness variation measured in the zone of apposition has been previously used to diagnose diaphragm paralysis. We assessed the feasibility and accuracy of this method to assess diaphragmatic function and its contribution to respiratory workload in critically ill patients under non-invasive ventilation.

METHODS

This was a preliminary physiological study in the intensive care unit of a university hospital. Twelve patients requiring planned non-invasive ventilation after extubation were studied while spontaneously breathing and during non-invasive ventilation at three levels of pressure support (5, 10 and 15 cmH(2)O). Diaphragm thickness was measured in the zone of apposition during tidal ventilation and the thickening fraction (TF) was calculated as (thickness at inspiration - thickness at expiration)/thickness at expiration. Diaphragmatic pressure-time product per breath (PTP(di)) was measured from oesophageal and gastric pressure recordings.

RESULTS

PTP(di) and TF both decreased as the level of pressure support increased. A significant correlation was found between PTP(di) and TF (ρ = 0.74, p < 0.001). The overall reproducibility of TF assessment was good but the coefficient of repeatability reached 18% for inter-observer reproducibility.

CONCLUSIONS

Ultrasonographic assessment of the diaphragm TF is a non-invasive method that may prove useful in evaluating diaphragmatic function and its contribution to respiratory workload in intensive care unit patients.

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.

BACKGROUND

Activation of coagulation and fibrinolysis play a role in the pathophysiology of experimental arthritis.

OBJECTIVE

To determine the extent of activation of the coagulation and fibrinolytic pathways in different joint diseases in humans and to ascertain the factors that may influence fibrin deposition within the joint.

METHODS

Plasma from normal subjects (controls, n= 21) and plasma and synovial fluid samples from patients with rheumatoid arthritis (RA; n = 64), osteoarthritis (OA; n = 29), spondyloarthropathy (SpA; n = 22) and crystal arthritis (CA; n = 25) were analyzed for the levels of TF (tissue factor) and tissue factor pathway inhibitor (TFPI) activities, thrombin-antithrombin III (TAT) complexes, and F1 + 2 (thrombin fragment), fibrin d-dimer and thrombin-activated fibrinolysis inhibitor (TAFI) antigenic levels. The measurements were analyzed by pairwise correlation with each other as well as with standard parameters of inflammation [C-reactive protein (CRP), joint leukocyte count]. Inter-group comparisons were performed to look for disease-specific differences.

RESULTS

Compared with healthy controls, patients with joint diseases had higher levels of TAT, F1 + 2 and d-dimers in their plasma. In the synovial fluid, TF activity, TAT, d-dimers, and TAFI were significantly higher in inflammatory arthritides than in OA. The levels were highest in RA patients. In the plasma, TF activity was correlated with TAT and d-dimer levels with CRP, TFPI, and TAT. In the synovial fluid, TF activity correlated with plasma CRP levels, synovial fluid leukocyte count, and synovial TAT and TAFI levels. In addition, synovial d-dimers correlated with CRP, and synovial TAFI levels were correlated with synovial F1 + 2 and TAT.

CONCLUSIONS

Activation of the coagulation and fibrinolytic cascades in the joint and in the circulation is evident in both inflammatory and degenerative joint diseases. Within the joint, inflammatory mechanisms leading to TF-mediated activation of the coagulation pathway and subsequent fibrin deposition is the most likely explanation for the observed findings. In the plasma, the link between inflammation (CRP increase) and TF activation is weak, and a non-TF-mediated mechanism of coagulation activation could explain these findings. RA is characterized by significantly higher levels of TAT in the synovial fluid and plasma than other arthritides. Although fibrinolytic activity is linked to inflammation, the increased amounts of TAFI in the joint, particularly in RA, may explain why fibrin formation is so prominent in this condition compared with other joint diseases.

Externalizing is a broad construct that reflects propensity toward a variety of impulse control problems, including antisocial personality disorder and substance use disorders. Two event-related potential responses known to be reduced among individuals high in externalizing proneness are the P300, which reflects postperceptual processing of a stimulus, and the error-related negativity (ERN), which indexes performance monitoring based on endogenous representations. In the current study, the authors used a simulated gambling task to examine the relation between externalizing proneness and the feedback-related negativity (FRN), a brain response that indexes performance monitoring related to exogenous cues, which is thought to be highly related to the ERN. Time-frequency (TF) analysis was used to disentangle the FRN from the accompanying P300 response to feedback cues by parsing the overall feedback-locked potential into distinctive theta (4-7 Hz) and delta (<3 Hz) TF components. Whereas delta-P300 amplitude was reduced among individuals high in externalizing proneness, theta-FRN response was unrelated to externalizing. These findings suggest that in contrast with previously reported deficits in endogenously based performance monitoring (as indexed by the ERN), individuals prone to externalizing problems show intact monitoring of exogenous cues (as indexed by the FRN). The results also contribute to a growing body of evidence indicating that the P300 is attenuated across a broad range of task conditions in high-externalizing individuals.

Tissue factor (TF) is the high-affinity receptor for plasma factors VII and VIIa. TF plays a role in normal hemostasis by initiating the cell-surface assembly and propagation of the coagulation protease cascade. Outside the vasculature, TF expression is highly dependent upon cell type. TF can also be induced by inflammatory mediators to appear on monocytes and vascular endothelial cells as a component of cellular immune responses. As an initial step toward elucidating the regulatory regions involved in control of TF gene expression, we have established the organization of the 12.4 kbp human TF gene and its complete DNA sequence. There are six exons separated by five introns. Within intron 5, we have mapped the single nucleotide difference which leads to the previously described MspI polymorphism; the same intron also contains an apparently polymorphic PstI site. The TF gene also contains three full-length Alu repeats and one partial Alu repeat. A single major transcription start site was identified 26 bp downstream from a TATA consensus promoter element. The putative promoter and first exon are located within a 1.2 kbp region of very high G + C content which fits the criteria of an HTF island. A cluster of predicted binding sites for a number of known transcription factors was found to coincide with this putative promoter region. These factors included AP-1 and AP-2 which can mediate the effects of phorbol esters, agonists known to induce TF expression in monocytes and vascular endothelial cells.

Knowing transcription factors (TFs) involved in the yeast cell cycle is helpful for understanding the regulation of yeast cell cycle genes. We therefore developed two methods for predicting (i) individual cell cycle TFs and (ii) synergistic TF pairs. The essential idea is that genes regulated by a cell cycle TF should have higher (lower, if it is a repressor) expression levels than genes not regulated by it during one or more phases of the cell cycle. This idea can also be used to identify synergistic interactions of TFs. Applying our methods to chromatin immunoprecipitation data and microarray data, we predict 50 cell cycle TFs and 80 synergistic TF pairs, including most known cell cycle TFs and synergistic TF pairs. Using these and published results, we describe the behaviors of 50 known or inferred cell cycle TFs in each cell cycle phase in terms of activation/repression and potential positive/negative interactions between TFs. In addition to the cell cycle, our methods are also applicable to other functions.

A simple continuum model of a de novo designed model of a four-helix bundle is presented. The thermodynamics and kinetics of the model are studied using Langevin simulations. We use a three-letter minimal off-lattice representation of a de novo designed four-helix bundle protein. The native state of the model, which can be thought of as an alpha-carbon representation of the peptide chain, is a caricature of the sequence designed by Ho and Degrado and shows several characteristics found in the naturally occurring four-helix bundles. These include the structural aspects and the relative stability of the native conformation. The model four-helix bundle shows two characteristic temperatures T theta and Tf. The former is the temperature above which the structure resembles that of the random coil. Below the first-order folding transition temperature Tf the chain adopts the native conformation corresponding to the four-helix bundle. It is shown that in order to obtain a unique native structure a proper free energy balance between secondary and tertiary interactions is needed. The thermal denaturation starting from the unique native conformation indicates that at least a three-state analysis is required. The intermediates in the equilibrium thermal denaturation are all found to be native-like. The kinetics of refolding starting from an ensemble of denatured states shows that the acquisition of the native conformation takes place via a kinetic partitioning mechanism. A fraction of molecules, phi, reaches the native state by a topology inducing nucleation collapse mechanism, while the remainder (1-phi) follows a complex three-stage multipathway process. We suggest, in accord with our earlier studies, that phi is essentially determined by the intrinsic temperature scales T theta and Tf. Our studies indicate that better design of proteins can be achieved by making T theta as close to Tf as possible. Experimental implications for de novo design of proteins are briefly discussed.

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

The TATA-binding proteins (TBP) from both human and Drosophila have been shown to exist in various distinct multiprotein complexes that are required, respectively, for transcription by all three RNA polymerases. In contrast, in vitro biochemical analyses have suggested that yeast TBP exists as a monomeric 27-kDa protein free in solution. We have examined the oligomerization state of yeast TBP and report here that yeast TBP, like human and Drosophila TBPs, is also stably associated with other proteins in vitro. Using anti-TBP antibodies we have immunopurified yeast TBP and associated factors (TBP-associated factors or TAFs). When this fraction was analyzed by SDS-polyacrylamide gel electrophoresis, polypeptides of approximate relative molecular size ranging from 170 to 60 kDa are prominently represented. Immunoblot analysis revealed that one of these TAFs, TAF70, corresponds to BRF1/TDS4/PCF4, a subunit of transcription factor (TF) IIIB. Furthermore, this highly purified TAF fraction can reconstitute polymerase III transcription when supplemented with purified RNA polymerase III and TFIIIC. Our data indicate that our TAF fraction contains TFIIIB transcription factor activity and that all the subunits of yeast TFIIIB are stably complexed with TBP.

The bilaterian animals are divided into three great branches: the Deuterostomia, Ecdysozoa, and Lophotrochozoa. The evolution of developmental mechanisms is less studied in the Lophotrochozoa than in the other two clades. We have studied the expression of Hox genes during larval development of two lophotrochozoans, the polychaete annelids Nereis virens and Platynereis dumerilii. As reported previously, the Hox cluster of N. virens consists of at least 11 genes (de Rosa R, Grenier JK, Andreeva T, Cook CE, Adoutte A, Akam M, Carroll SB, Balavoine G, Nature, 399:772-776, 1999; Andreeva TF, Cook C, Korchagina NM, Akam M, Dondua AK, Ontogenez 32:225-233, 2001); we have also cloned nine Hox genes of P. dumerilii. Hox genes are mainly expressed in the descendants of the 2d blastomere, which form the integument of segments, ventral neural ganglia, pre-pygidial growth zone, and the pygidial lobe. Patterns of expression are similar for orthologous genes of both nereids. In Nereis, Hox2, and Hox3 are activated before the blastopore closure, while Hox1 and Hox4 are activated just after this. Hox5 and Post2 are first active during the metatrochophore stage, and Hox7, Lox4, and Lox2 at the late nectochaete stage only. During larval stages, Hox genes are expressed in staggered domains in the developing segments and pygidial lobe. The pattern of expression of Hox cluster genes suggests their involvement in the vectorial regionalization of the larval body along the antero-posterior axis. Hox gene expression in nereids conforms to the canonical patterns postulated for the two other evolutionary branches of the Bilateria, the Ecdysozoa and the Deuterostomia, thus supporting the evolutionary conservatism of the function of Hox genes in development.

The determination of inulin concentration in nanoliter fluid samples is fundamental to micropuncture investigations of renal function, and this is generally accomplished through the use of radioisotopes. We report here a simple and reliable alternative to the use of radioisotopes that employs FITC-labeled inulin. Samples containing FITC-inulin are stored between oil columns in constant-bore microcapillary tubes, which are then used as cuvettes to determine fluorescence on a microscope fluorometer. Standard curves were generated and found to be linear, with correlation coefficients (R) exceeding 0.99 in every case. Although the fluorescence of FITC-inulin was found to be pH dependent, the pH and fluorescence of each 20- to 40-nl sample could be normalized by the addition of 1 nl of 0.5 M HEPES at pH 7.5. In mice prepared for standard micropuncture, simultaneous measurements of tubular fluid-to-plasma ratios (TF/P) using FITC-inulin and [125I]iothalamate were highly correlated (slope = 0.95, y-intercept = 0.01, R = 0.942), as were whole kidney measurements of glomerular filtration rate (GFR) (slope = 1.25, y-intercept = -53.5 microliter/min, R = 0.99). Micropuncture determinations of late-proximal samples from mice before and after treatment with acetazolamide showed expected changes: TF/P of FITC-inulin decreased from 1.89 +/- 0.07 to 1.48 +/- 0.10; single-nephron GFR (SNGFR) decreased from 9.64 +/- 1.1 to 6.65 +/- 1. 0 nl/min; and fractional fluid reabsorption decreased from 45.3 +/- 1.9 to 26.8 +/- 5.2%. Measurements of TF/P of FITC-inulin, volume, and SNGFR using this technique were stable for at least 2 wk when samples were stored in the dark at 4 degreesC. These data demonstrate that this simple method for determining inulin clearance represents a viable and accurate alternative to radioactive methods. This approach has the added benefits of being relatively inexpensive and leaving the micropuncture sample intact.