Blood group-related antigens have been attractive targets for immunotherapy of cancer since their initial identification as cancer-related antigens. However, available information on the relative expression of most of these antigens on human malignant and normal tissues has been insufficient for selecting optimal antigens and tumors for immune attack. In this study, the distribution of the blood group-related antigens TF, Tn, sTn, Le(a), sialyl Le(a), Le(b), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on 13 types of cancer and 16 normal tissues was compared. Our results show that sTn is strongly expressed on cancers of breast, colon, stomach, ovary, prostate and uterus; Tn on prostate cancer; TF on cancers of breast, colon, ovary, prostate and uterus; Le(y) on the cancers of colon, lung, pancreas and ovary; Le(a) and Le(x) on gastric cancer; and sialyl Le(a) and sialyl Le(x) on colon cancer. The complete absence of these antigens on cancers of neuroectodermal or mesodermal origin including melanoma, sarcoma, neuroblastoma and B cell lymphoma is as striking as their widespread presence on tumors of epithelial origin. Normal tissues were also tested. Tn and Le(b) were only detected on gastric and ovarian epithelia; sTn on Leydig cells of testis in addition to gastric and ovarian epithelia; Le(x) and sialyl Le(x) on polymorphonuclear leukocytes; and TF, Le(a), sialyl Le(a), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on epithelia from a variety of tissues.

OBJECTIVE

miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML), namely, DNMTs, CDK6, SP1, KIT, and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and better outcome in AML. Thus, designing a strategy to increase miR-29b levels in AML blasts may be of therapeutic value. However, free synthetic miRs are easily degraded in bio-fluids and have limited cellular uptake. To overcome these limitations, we developed a novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf-NP-miR-29b).

METHODS

Delivery efficiency was investigated by flow cytometry, confocal microscopy, and quantitative PCR. The expression of miR-29b targets was measured by immunoblotting. The antileukemic activity of Tf-NP-miR-29b was evaluated by measuring cell proliferation and colony formation ability and in a leukemia mouse model.

RESULTS

Tf-NP-miR-29b treatment resulted in more than 200-fold increase of mature miR-29b compared with free miR-29b and was approximately twice as efficient as treatment with non-transferrin-conjugated NP-miR-29b. Tf-NP-miR-29b treatment significantly downregulated DNMTs, CDK6, SP1, KIT, and FLT3 and decreased AML cell growth by 30% to 50% and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf-NP-miR-29b had significantly longer survival compared with Tf-NP-scramble (P = 0.015) or free miR-29b (P = 0.003). Furthermore, priming AML cell with Tf-NP-miR-29b before treatment with decitabine resulted in marked decrease in cell viability in vitro and showed improved antileukemic activity compared with decitabine alone (P = 0.001) in vivo.

CONCLUSIONS

Tf-NP effectively delivered functional miR-29b, resulting in target downregulation and antileukemic activity and warrants further investigation as a novel therapeutic approach in AML.

Neuroanatomical studies in macaque monkeys have demonstrated that the perirhinal and parahippocampal (PRPH) cortices are strongly interconnected with the hippocampal formation. Recent behavioral evidence indicates that these cortical regions are importantly involved in normal recognition memory function. The PRPH cortices are also interconnected with the amygdaloid complex, although comparatively little is known about the precise topography of these connections. We investigated the topographic organization of reciprocal connections between the amygdala and the PRPH cortices by placing anterograde and retrograde tracers throughout these three regions. We found that there was an organized arrangement of connections between the amygdala and the PRPH cortices and that the deep (lateral, basal, and accessory basal) nuclei of the amygdaloid complex were the source of most connections between the amygdala and the PRPH cortices. The temporal polar regions of the perirhinal cortex had the strongest and most widespread interconnections with the amygdala. Connections from more caudal levels of the perirhinal cortex had a more discrete pattern of termination. Perirhinal inputs to the amygdala terminated primarily in the lateral nucleus, the magnocellular and parvicellular divisions of the basal nucleus, and the magnocellular division of the accessory basal nucleus. Return projections originated predominately in the lateral nucleus, the intermediate and parvicellular divisions of the basal nucleus, and the magnocellular division of the accessory basal nucleus. The interconnections between the amygdala and the parahippocampal cortex were substantially less robust than those with the perirhinal cortex and mainly involved the basal nucleus. Area TF was more strongly interconnected with the amygdala than was area TH. Input from the parahippocampal cortex terminated predominantly in the lateral half of the parvicellular division of the basal nucleus but also to a lesser extent in the magnocellular division of the basal nucleus and the lateral nucleus. Return projections originated predominantly in the magnocellular division of the basal nucleus and were directed almost exclusively to area TF.

BACKGROUND

Formation of tumour cell aggregation/emboli prolongs the survival of circulating tumour cells in the circulation, enhances their physical trapping in the micro-vasculature and thus increases metastatic spread of the cancer cells to remote sites.

RESULTS

It shows here that the presence of the galactoside-binding galectin-3, whose concentration is markedly increased in the blood circulation of cancer patients, increases cancer cell homotypic aggregation under anchorage-independent conditions by interaction with the oncofetal Thomsen-Friedenreich carbohydrate (Galbeta1,3GalNAcalpha-, TF) antigen on the cancer-associated transmembrane mucin protein MUC1. The galectin-3-MUC1 interaction induces MUC1 cell surface polarization and exposure of the cell surface adhesion molecules including E-cadherin. The enhanced cancer cell homotypic aggregation by galectin-MUC1 interaction increases the survival of the tumour cells under anchorage-independent conditions by allowing them to avoid initiation of anoikis (suspension-induced apoptosis).

CONCLUSIONS

These results suggest that the interaction between free circulating galectin-3 and cancer-associated MUC1 promotes embolus formation and survival of disseminating tumour cells in the circulation. This provides new information into our understanding of the molecular mechanisms of cancer cell haematogenous dissemination and suggests that targeting the interaction of circulating galectin-3 with MUC1 in the circulation may represent an effective therapeutic approach for preventing metastasis.

OBJECTIVE

Recently, large intergenic non-coding ribonucleic acids-RoR (linc-RoR) was reported to regulate expression of core stem cell transcription factors (TFs), but its role in endometrial tumorsphere is still unknown.

METHODS

Fluorescence in situ hybridization (FISH) was used to characterize linc-RoR expression in ETs. After construction of adenovirus vectors carrying green fluorescent protein (GFP), these vectors were transfected into ETs to estimate the effects of overexpression or knocked down expression of miR-145, linc-RoR or Dicer. Flow cytometry was employed to ascertain transfection efficiency, and real-time polymerase chain reaction (RT-PCR) was employed to compare their levels. Colony formation was analyzed using cultured gelatin-coated tissue cultures. miR-145 potential targeting sites in linc-RoR were mutated using a site-directed mutagenesis kit to verify its competing endogenous RNA (ceRNA) effects.

RESULTS

Expression of linc-RoR and core stem cell TFs was associated with the pluripotent state of ETs, whereas miR-145 expression increased after ET differentiation. Greater expression of miR-145 could lead to down-regulation of linc-RoR and core TFs, and decreased colony formation. Converse effects could be achieved after knocked-down miR-145 expression. The effects of miR-145 could be eliminated after increasing the expression of linc-RoR in ETs or mutated targeted sequences in linc-RoR. Knocked-down Dicer expression could improve the expression of linc-RoR and core TFs.

CONCLUSIONS

Linc-RoR is a ceRNA and acts as a miR-145 "sponge" to inhibit mediation of the differentiation of ETs by miR-145. These results suggest that linc-RoR has an important role during endometrial carcinogenesis.

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) allows researchers to determine the genome-wide binding locations of individual transcription factors (TFs) at high resolution. This information can be interrogated to study various aspects of TF behaviour, including the mechanisms that control TF binding. Physical interaction between TFs comprises one important aspect of TF binding in eukaryotes, mediating tissue-specific gene expression. We have developed an algorithm, spaced motif analysis (SpaMo), which is able to infer physical interactions between the given TF and TFs bound at neighbouring sites at the DNA interface. The algorithm predicts TF interactions in half of the ChIP-seq data sets we test, with the majority of these predictions supported by direct evidence from the literature or evidence of homodimerization. High resolution motif spacing information obtained by this method can facilitate an improved understanding of individual TF complex structures. SpaMo can assist researchers in extracting maximum information relating to binding mechanisms from their TF ChIP-seq data. SpaMo is available for download and interactive use as part of the MEME Suite (http://meme.nbcr.net).

IL-11 is a novel cytokine with a variety of biofunctions which overlap with those of IL-6. We have previously identified IL-11 specific binding protein which is distinct from that of IL-6 in a number of cell lines. The similarities in biofunctions and differences in ligand binding proteins between IL-11 and IL-6 prompted us to investigate whether IL-11 shares common signal transduction mechanisms with IL-6. We have examined early signals triggered by IL-11 or IL-6 in a multifactor-dependent human erythroleukemic cell line, TF-1. The results showed that IL-11 and IL-6 can both stimulate cell proliferation, induce similar pattern of protein tyrosine phosphorylation, and activate the same proto-oncogene (junB) expression in TF-1 cells. These findings imply that IL-11 and IL-6 share similar early signaling events with the possibility of using the same signal transducer, gp130. We next tested whether IL-11 induced signaling can be inhibited by anti-gp130 antibodies which blocked IL-6-mediated functions. It was observed that anti-gp130 antibodies abolished cell proliferation, protein tyrosine phosphorylation, and junB gene expression elicited by IL-11 or IL-6 in TF-1 cells. The same antibodies, however, had no effect on granulocyte-macrophage colony stimulating factor or erythropoietin-induced TF-1 cell proliferation. Finally, anti-IL-6R antibody inhibited the ability of IL-6, but not IL-11, to transduce early signals in TF-1 cells. These results demonstrate that IL-11 and IL-6 utilize different ligand binding proteins, but share common signal transducer, gp130, in TF-1 cells.

BACKGROUND

Transcription Factors (TFs) and microRNAs (miRNAs) are key players for gene expression regulation in higher eukaryotes. In the last years, a large amount of bioinformatic studies were devoted to the elucidation of transcriptional and post-transcriptional (mostly miRNA-mediated) regulatory interactions, but little is known about the interplay between them.

METHODS

Here we describe a dynamic web-accessible database, CircuitsDB, supporting a genome-wide transcriptional and post-transcriptional regulatory network integration, for the human and mouse genomes, based on a bioinformatic sequence-analysis approach. In particular, CircuitsDB is currently focused on the study of mixed miRNA/TF Feed-Forward regulatory Loops (FFLs), i.e. elementary circuits in which a master TF regulates an miRNA and together with it a set of Joint Target protein-coding genes. The database was constructed using an ab-initio oligo analysis procedure for the identification of the transcriptional and post-transcriptional interactions. Several external sources of information were then pooled together to obtain the functional annotation of the proposed interactions. Results for human and mouse genomes are presented in an integrated web tool, that allows users to explore the circuits, investigate their sequence and functional properties and thus suggest possible biological experiments.

CONCLUSIONS

We present CircuitsDB, a web-server devoted to the study of human and mouse mixed miRNA/TF Feed-Forward regulatory circuits, freely available at: http://biocluster.di.unito.it/circuits/

The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, we have used a combinatorial strategy to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yields a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide.

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (TfTf). TfTf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of TfTfTf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained TfTfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with TfTfTf. Retention of TfTfR since TfTfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by TfTf.

Naturally occurring t haplotypes are chromosome 17 polymorphisms that suppress genetic recombination in t/+ heterozygotes over a long distance that includes the H-2 complex. There is strong linkage disequilibrium between t haplotypes and H-2 haplotypes; over 20 independently isolated t chromosomes representing eight different complementation groups share only four H-2 haplotypes. Thus t haplotypes and their associated H-2 loci are inherited en bloc as a "supergene" complex, whose frequency is driven in wild mouse populations by their high transmission from male t heterozygotes. This phenomenon must therefore serve as an important regulator of H-2 polymorphisms. Genes within the region of recombination suppression in t haplotypes have been mapped by crossing-over that occurs readily between two different t haplotypes situated in trans, and by this means we show here that the H-2 complex occupies an anomalous position in t haplotypes, mapping proximal to the locus of tf closely flanked by t-lethal mutations.

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse failed to release TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.

To further elucidate the mechanism of transcriptional initiation, we used synthetic oligonucleotides to prepare templates containing heteroduplex regions of varying size and location along the DNA of the adenovirus major late promoter. Unlike closed, linear DNA, or DNA with a downstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcription factor (TF) IIB to direct specific and accurate initiation in vitro by calf thymus RNA polymerase II. In the presence of TFIIF, initiation was possible on closed, linear DNA, but an upstream mismatch region still stimulated transcriptional initiation by more than 100-fold, leading to production of approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or ATP. The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a mismatch did not require that the initiation site be included in the heteroduplex region. Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was also achieved when a mismatch region was introduced from -9 to +3. Our results suggest that initiation by RNA polymerase II in the absence of transcriptional activation is limited by melting of the promoter DNA upstream of the initiation site.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite down-regulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the iron-responsive elements found in the 3' untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE -/-). Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not down-regulated with dietary iron loading or in the HFE -/- model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH.

We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites.

APETALA2 (AP2) transcription factors (TFs) play very important roles in plant growth and development and in defense response. Here, we report functional characterization of four AP2 TF family genes [(RAP2.6 (At1g43160), RAP2.6L (At5g13330), DREB 26 (At1g21910) and DREB19 (At2g38340)] that were identified among NaCl inducible transcripts in abscisic acid responsive 17 (ABR17) transgenic Arabidopsis in our previous microarray analyses. DREB19 and DREB26 function as transactivators and localize in the nucleus. All four genes were abundant in early vegetative and flowering stages, although the magnitude of the expression varied. We observed tissue specific expression patterns for RAP2.6, RAP2.6L, DREB19 and DREB26 in flowers and other organs. RAP2.6 and RAP2.6L were responsive to stress hormones like jasmonic acid, salicylic acid, abscisic acid and ethylene in addition to salt and drought. DREB19 and DREB26 were less responsive to stress hormones, but the former was highly responsive to salt, heat and drought. Overexpression of RAP2.6 in Arabidopsis resulted in a dwarf phenotype with extensive secondary branching and small siliques, and DREB26 overexpression resulted in deformed plants. However, overexpression of RAP2.6L and DREB19 enhanced performance under salt and drought stresses without affecting phenotype. In summary, we have demonstrated that RAP2.6, RAP2.6L, DREB26 and DREB19 are transactivators, they exhibit tissue specific expression, and they participate in plant developmental processes as well as biotic and/or abiotic stress signaling. It is possible that the results from this study on these transcription factors, in particular RAP2.6L and DREB19, can be utilized in developing salt and drought tolerant plants in the future.

Speech reception thresholds (SRTs) were measured with a competing talker background for signals processed to contain variable amounts of temporal fine structure (TFS) information, using nine normal-hearing and nine hearing-impaired subjects. Signals (speech and background talker) were bandpass filtered into channels. Channel signals for channel numbers above a "cut-off channel" (CO) were vocoded to remove TFS information, while channel signals for channel numbers of CO and below were left unprocessed. Signals from all channels were combined. As a group, hearing-impaired subjects benefited less than normal-hearing subjects from the additional TFS information that was available as CO increased. The amount of benefit varied between hearing-impaired individuals, with some showing no improvement in SRT and one showing an improvement similar to that for normal-hearing subjects. The reduced ability to take advantage of TFS information in speech may partially explain why subjects with cochlear hearing loss get less benefit from listening in a fluctuating background than normal-hearing subjects. TFS information may be important in identifying the temporal "dips" in such a background.

It is becoming increasingly apparent that the causes of autism spectrum disorders (ASD) are due to both genetic and environmental factors. Animal studies provide important translational models for elucidating specific genetic or environmental factors that contribute to ASD-related behavioral deficits. For example, mouse research has demonstrated a link between maternal immune activation and the expression of ASD-like behaviors. Although these studies have provided insights into the potential causes of ASD, they are limited in their ability to model the important interactions between genetic variability and environmental insults. This is of particular concern given the broad spectrum of severity observed in the human population, suggesting that subpopulations may be more susceptible to the adverse effects of particular environmental insults. It is hypothesized that the severity of effects of maternal immune activation on ASD-like phenotypes is influenced by the genetic background in mice. To test this, pregnant dams of two inbred strains (that is, C57BL/6J and BTBR T(+)tf/J) were exposed to the viral mimic polyinosinic-polycytidylic acid (polyI:C), and their offspring were tested for the presence and severity of ASD-like behaviors. To identify differences in immune system regulation, spleens were processed and measured for alterations in induced cytokine responses. Strain-treatment interactions were observed in social approach, ultrasonic vocalization, repetitive grooming and marble burying behaviors. Interestingly, persistent dysregulation of adaptive immune system function was only observed in BTBR mice. Data suggest that behavioral and immunological effects of maternal immune activation are strain-dependent in mice.

As part of a long-term study designed to examine the ontogeny of visual memory in monkeys and its underlying neural circuitry, we have examined the connections between inferior temporal cortex and medial temporal-lobe structures in infant and adult monkeys. Inferior temporal cortical areas TEO and TE were injected with WGA conjugated to HRP and tritiated amino acids, respectively, or vice versa, in 1-week-old and 3-4-yr-old Macaca mulatta, and the distributions of labeled cells and terminals were examined in both limbic structures and temporal-lobe cortical areas. In adult monkeys, inferior temporal-limbic connections included projections from area TEO to the dorsal portion of the lateral nucleus of the amygdala and from area TE to the lateral and lateral basal nuclei; inputs to both areas TEO and TE included those from the lateral, lateral basal, and medial basal nuclei of the amygdala and to area TE from the accessory basal nucleus. Additional limbic inputs to both areas TEO and TE arose from the posterior portion of the presubiculum. In infant monkeys, we found, in addition to these adultlike connections, a projection from area TEO to the lateral basal nucleus of the amygdala. Inferior temporal cortical connections in adult monkeys included projections from area TEO to area TE and, in turn, from area TE to area TG and perirhinal area 36, as well as from area TE back to area TEO; inputs to both areas TEO and TE included those from area TG, perirhinal areas 35 and 36, and parahippocampal areas TF and TH. All of these adultlike connections were also observed in infant monkeys, but, in addition, the infants showed projections from area TE to perirhinal area 35 as well as to parahippocampal areas TF and TH, and from area TEO to area TF. Moreover, in infants, the projection from area TE to perirhinal area 36 was considerably more widespread than in adults, both in areal extent and in laminar distribution. The results therefore indicate the existence of projections in infant monkeys from inferior temporal areas to the amygdala, perirhinal cortex, and parahippocampal cortex that are either totally eliminated in adults or more refined in their distribution. Both elimination and refinement of projections thus appear to characterize the maturation of axonal pathways between the inferior temporal cortex and medial temporal-lobe structures in monkeys.

We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P < .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors that belong to the steroid/thyroid hormone receptor (TR) superfamily and can repress the transcriptional activity of several target genes; however, the precise mechanism of this repression is unknown. Transfection of a Gal4 DNA-binding domain fused to the putative ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly represses the basal transcriptional activity of a reporter gene containing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TFI delta35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that the repressor domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa cells. In addition, increasing concentrations of TRbeta also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI delta35, can squelch the silencing activity of the unliganded TRbeta. Collectively, these results indicate that COUP-TFI and TRbeta share a common corepressor(s) for their silencing activity. To determine which corepressor is involved in the COUP-TF-silencing activity, we used a yeast two-hybrid and in vitro GST pull-down assays to demonstrate that COUP-TFI can interact with the fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids 921-2453 and the fragments of SMRT (silencing mediator for retinoic acid receptor and TR) encoding amino acids 29-564 and 565-1289, respectively. Interestingly, the fragment of SMRT encoding amino acids 1192-1495, which strongly interacts with TRbeta, interacts very weakly with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates the silencing activity of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, our studies indicate that N-CoR and SMRT act as corepressors for the COUP-TFI silencing activity.

Thrombosis occurs in a dynamic rheological field that constantly changes as the thrombus grows to occlusive dimensions. In the initiation of thrombosis, flow conditions near the vessel wall regulate how quickly reactive components are delivered to the injured site and how rapidly the reaction products are disseminated. Whereas the delivery and removal of soluble coagulation factors to the vessel is thought to occur via classic convection-diffusion phenomena, the movement of cells and platelets to the injured wall is strongly augmented by flow-dependent cell-cell collisions that enhance their ability to interact with the wall. In addition, increased shear conditions have been shown to activate platelets, alter the cellular localization of proteins such as tissue factor (TF) and TF pathway inhibitor, and regulate gene production. In the absence of high shearing forces, red cells, leukocytes, and platelets can form stable aggregates with each other or cells lining the vessel wall, which, in addition to altering the biochemical makeup of the aggregate or vessel wall, effectively increases the local blood viscosity. Thus, hemodynamic forces not only regulate the predilection of specific anatomic sites to thrombosis, but they strongly influence the biochemical makeup of thrombi and the reaction pathways involved in thrombus formation.

Frequency selectivity, temporal fine-structure (TFS) processing, and speech reception were assessed for six normal-hearing (NH) listeners, ten sensorineurally hearing-impaired (HI) listeners with similar high-frequency losses, and two listeners with an obscure dysfunction (OD). TFS processing was investigated at low frequencies in regions of normal hearing, through measurements of binaural masked detection, tone lateralization, and monaural frequency modulation (FM) detection. Lateralization and FM detection thresholds were measured in quiet and in background noise. Speech reception thresholds were obtained for full-spectrum and lowpass-filtered sentences with different interferers. Both the HI listeners and the OD listeners showed poorer performance than the NH listeners in terms of frequency selectivity, TFS processing, and speech reception. While a correlation was observed between the monaural and binaural TFS-processing deficits in the HI listeners, no relation was found between TFS processing and frequency selectivity. The effect of noise on TFS processing was not larger for the HI listeners than for the NH listeners. Finally, TFS-processing performance was correlated with speech reception in a two-talker background and lateralized noise, but not in amplitude-modulated noise. The results provide constraints for future models of impaired auditory signal processing.

Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.