microRNA-199a-5p protects hepatocytes from bile acid-induced sustained endoplasmic reticulum stress.

Sustained endoplasmic reticulum (ER) stress has been linked to cell death and the pathogenesis of many liver diseases, including toxic liver, cholestasis, and infectious liver disease. The cellular pathways that attenuate hepatic ER stress have been the focus of many recent studies, but the role of microRNAs (miRNA) in this process remains unknown. Here, we report that one of the most abundant miRNAs in hepatocytes, miR-199a-5p, was elevated in both bile acid- and thapsigargin (TG)-stimulated cultured hepatocytes, as well as in the liver of bile duct-ligated mice. We identify the misfolded protein chaperone GRP78, as well as the unfolded protein response transducers endoplasmic reticulum to nucleus signaling 1 and activating transcription factor 6 as direct targets of miR-199a-5p, and show that endogenous miR-199a-5p represses the 3' untranslated regions (UTRs) of their mRNAs. Through gain-of-function and loss of function approaches, we demonstrate that the elevated miR-199-5p disrupts sustained ER stress and prevents hepatocytes from undergoing bile acid- or TG-induced cell death. Furthermore, we reveal that the transcription factor AP-1 is a strong positive regulator of miR-199a-5p. In brief, our study demonstrates that AP-1/miR-199a-5p and ER stress mediators form a feedback loop, which shields hepatocytes from sustained ER stress and protects the liver from injury. On the basis of these findings, we also suggest that the miRNA miR-199a-5p is a potential target for clinical approaches aiming to protect hepatocytes in liver disease.

Adjuvant transcatheter arterial chemoembolization for intrahepatic cholangiocarcinoma after curative surgery: retrospective control study.

BACKGROUND: Effects of adjuvant transcatheter arterial chemoembolization (TACE) for intrahepatic cholangiocarcinoma (ICC) radical surgery have never been evaluated. METHODS: A retrospective analysis was conducted on 125 ICC patients who had undergone operations with curative intent in Shanghai Eastern Hepatobiliary Surgery Hospital from July 2002 to December 2003. Of these patients, 53 underwent adjuvant TACE (TACE group) and 72 did not (non-TACE group). Adjuvant TACE was performed one time 1.5-2.0 months after the operation. RESULTS: Follow-up was performed at a median of 18 months (range 3-96 months). There was no significant recurrence-free survival (RFS) difference between the TACE and non-TACE groups (P = 0.659). The 1-, 3-, and 5-year overall survival (OS) rates were 69.8, 37.7, and 28.3%, respectively, for the TACE group and 54.2, 25.0, and 20.8%, respectively, for the non-TACE group (P = 0.045). Among 54 patients with a recurrence time of ≤ 3 months, the OS rate of the TACE group was better than that of the non-TACE group (P < 0.001). For 59 patients with a recurrence time later than the median RFS, no significant RFS difference was found between the TACE and non-TACE groups (P = 0.681). These results indicate that TACE could not delay recurrence but could prolong the OS of patients with early recurrence. CONCLUSIONS: Adjuvant TACE after radical surgery was associated with better survival among the ICC patients with early recurrence.

Cloning and RNAi-mediated functional characterization of MaLac2 of the pine sawyer, Monochamus alternatus.

Laccase, a member of a group of proteins collectively known as multicopper oxidases, is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. Laccase 2 has been proved as the gene required for beetle cuticle tanning through RNA interference (RNAi) experiments on red flour beetle Tribolium castaneum. The pine sawyer beetle, Monochamus alternatus (Coleoptero: Cerambycidae) is the insect serving as a major vector of the pinewood nematode, Bursaphelenchus xylophilus, which is the causative agent for pine wilt disease. The cDNA of MaLac2 was cloned from the insect in this study. The conceptual amino-acid sequence deduced was much conserved with other known insect laccases, particularly with the enzyme of Tribolium castaneum. Injection in hemolymph of pine sawyer larva of dsRNA targeting the laccase 2 mRNA leads to important alterations of the tanning, hardening and sclerotization of the pupal and adult cuticles. Defaults appear in a dose-dependent manner and high loads of dsRNA are lethal. The decrease of the endogenous laccase 2 mRNA affects the procuticle which is thinner and without the characteristic piling up of successive layers. The observations reinforce the role of laccase 2 as an essential phenoloxidase for making cuticle.

The HOX homeodomain proteins block CBP histone acetyltransferase activity.

Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

Changes in HOXB6 homeodomain protein structure and localization during human epidermal development and differentiation.

HOX homeodomain proteins are master developmental regulators, which are now thought to function as transcription factors by forming cooperative DNA binding complexes with PBX or other protein partners. Although PBX proteins exhibit regulated subcellular localization and function in the nucleus in other tissues, little data exists on HOX and PBX protein localization during skin development. We now show that the HOXB6 protein is expressed in the suprabasal layer of the early developing epidermis and throughout the upper layers of late fetal and adult human skin. HOXB6 signal is cytoplasmic throughout fetal epidermal development, but substantially nuclear in normal adult skin. HOXB6 protein is also partially nuclear in hyperproliferative skin conditions, but appears to be cytoplasmic in basal and squamous cell carcinomas. Although all three PBX genes are expressed in fetal epidermis, none of the three PBX proteins exhibit nuclear co-localization with HOXB6 in either fetal or adult epidermis. RNA and protein data suggest that a truncated HOXB6 protein, lacking the homeodomain, is expressed in undifferentiated keratinocytes and that the full-length protein is induced by differentiation. GFP-fusion proteins were used to demonstrate that the full-length HOXB6 protein is localized to the nucleus while the truncated protein is largely cytoplasmic. Taken together, these data suggest that during epidermal development the truncated HOXB6 isoform may function by a mechanism other than as DNA binding protein, and that most of the nuclear, homeodomain-containing HOXB6 protein does not utilize PBX proteins as DNA binding partners in the skin. Published 2000 Wiley-Liss, Inc.

Functional cloning and characterization of a novel nonhomeodomain protein that inhibits the binding of PBX1-HOX complexes to DNA.

PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes. A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomain-containing protein that interacts with PBX1 as well as PBX2 and PBX3. RNA analysis revealed it to be expressed in CD34(+) hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues. The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins. This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus. The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences. Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences. Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX. Together these findings suggest that HPIP is a new regulator of PBX function.

HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells.

Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.

The Abd-B-like Hox homeodomain proteins can be subdivided by the ability to form complexes with Pbx1a on a novel DNA target.

Previous studies showed that the Hox homeodomain proteins from paralog groups 1-8 display cooperative DNA binding with the non-Hox homeodomain protein Pbx, mediated by a canonical YPWM. Although the Abd-B-like Hox proteins in paralogs 9-13 lack this sequence, Hoxb-9 and Hoxa-10 were reported to bind with Pbx1a to DNA. We show that these interactions require a tryptophan 6 amino acids N-terminal to the homeodomain. Binding site selection for Hoxb-9 with Pbx1a yielded ATGATTTACGAC, containing a novel TTAC Hox-binding site adjacent to a Pbx site. In the presence of Pbx1a, Hoxb-9 and Hoxa-10 bound to targets containing either TTAC or TTAT. These data extend previous findings that interactions with Pbx define a Hox protein binding code for different DNA sequences across paralog groups 1 through 10. Members of the 11, 12, and 13 paralogs do not cooperatively bind DNA with Pbx1a, despite the presence of tryptophan residues N-terminal to the homeodomain in Hoxd-12 and Hoxd-13. Hoxa-11, Hoxd-12, or Hoxd-13, in the presence of Pbx1a, selected a TTAC Hox site but lacking a Pbx1a site. These data suggest that Abd-B-like Hox proteins bind to a novel TTAC site and can be divided by their cooperative binding to DNA with Pbx1a.

[Can Doppler echocardiography help to avoid cardiac catheterization in the surgical decision-making in isolated left heart valve diseases?]. / L'échocardiographie Doppler peut-elle éviter le cathétérisme cardiaque dans l'indication opératoire des valvulopathies isolées du coeur gauche?

The aim of this study was to assess the value of non-invasive investigation based on clinical evaluation and Doppler echography in deciding the operative indications of patients with isolated left heart valvular lesions compared. Three hundred and thirty five patients were included in a prospective study: 78 had MR, 57 had AR, 150 had AS and 50 had MS. All underwent clinical. Doppler echography and catheter studies. The therapeutic decision was taken blind by two groups of 2 cardiologists. Group I took its decision based on clinical findings and results of Doppler echography whilst Group II took its decision on the clinical and catheter data. For each patient, one of the following three choices was proposed: 1) medical treatment: 2) surgery or valvuloplasty with balloon catheter; 3) request for further information. In addition, in group I, the need for coronary angiography was left to the appreciation of two cardiologists. The quantification of the valvular disease was concordant for groups I and II in 93, 97, 98.5 and 100% for MR, AR, AS and MS respectively. These percentages were respectively 97, 95, 92 and 100% for assessment of left ventricular function. The theoretical management decision was concordant between the two groups for 97% of MR, 94.7% of AR, 95.3% of AS and 94% of MS. Complementary information requiring invasive studies was required by group I in 3.9% of cases. A discordant opinion was obtained in 0.6% of cases (2 cases of AS). Coronary angiography was requested by the cardiologists of Group I in 34% of patients, identifying all patients who underwent coronary bypass surgery. These results show that cardiac catheterisation is no longer an essential diagnostic procedure for discussing the indications of valvular surgery in the majority of patients with isolated left heart lesions.

Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus.

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.

Clinical significance of intracavitary spontaneous echo contrast in patients with dilated cardiomyopathy.

To assess the occurrence rate and major determinants of spontaneous echo contrast and to examine its impact on thromboembolic events and mortality in patients with dilated cardiomyopathy, 86 hospitalized patients (73 men and 13 women, mean age 63 +/- 11 years) with dilated cardiomyopathy who underwent transthoracic and transesophageal echocardiographic examinations were followed up for a mean of 20 +/- 13 months. Spontaneous echo contrast was observed in 36 patients (42%) and was detected only with the transesophageal approach. It was seen in the left atrium in 33 patients, in both right and left atria in 1 patient, in both left atrium and left ventricle in 1 patient, and in the descending aorta in 1 patient. Spontaneous echo contrast was more frequent in the presence of atrial fibrillation (p < 0.05), left atrial enlargement (p < 0.02) and severely depressed left ventricular function (p < 0.01), but was less common in patients with moderate to severe mitral regurgitation (p < 0.05). This imaging phenomenon was the only significant independent predictor of intracardiac thrombus formation and previous and subsequent thromboembolic events. During follow-up, there were 26 deaths, and survival in patients with spontaneous echo contrast was significantly lower than in those without it (p < 0.02). A spontaneous echo contrast is commonly detected with transesophageal echocardiography in patients with dilated cardiomyopathy especially in the presence of atrial fibrillation, left atrial enlargement and severe left ventricular dysfunction. This imaging phenomenon represents an important marker for thromboembolic risk and may influence the treatment and clinical outcome of these patients.

Comparative value of Doppler echocardiography and cardiac catheterization for management decision-making in patients with left-sided valvular regurgitation.

OBJECTIVE: The purpose of this study was to examine the value of non-invasive clinical and Doppler echocardiographic findings, compared to cardiac catheterization, in management decision-making for patients with left-sided valvular regurgitation. METHODS: One hundred and thirty-five consecutive patients with left-sided valvular regurgitation who underwent cardiac catheterization and detailed Doppler echocardiography were prospectively studied. Two independent groups of experienced cardiologists, given clinical information combined with either Doppler echocardiographic or cardiac catheterization data, decided to operate, not to operate, or remained uncertain. RESULTS: In 63 (81%) of 78 patients with mitral regurgitation, there was agreement on the decision for valve surgery or medical treatment between Doppler echocardiography and cardiac catheterization. Valve repair was performed in 22 patients, which agreed with the echocardiographic decision. In the remaining 15 patients, although the severity and type of mitral valve lesions and left ventricular functional status were confirmed by Doppler echocardiography, the clinical decision was uncertain; additional information concerning coronary anatomy (13 patients) and pulmonary artery pressure (one patient) or both (one patient) was required. In 47 of 57 patients (82%) with aortic regurgitation, there was agreement on their management as a result of Doppler echocardiography and cardiac catheterization findings. In 10 patients, the clinical decision reached with the help of Doppler echocardiography alone was uncertain and coronary (seven patients), left ventricular (two patients) angiography or aortography (one patient) were requested. Overall, there were no conflicting clinical decisions made by the two methods in patients with either mitral or aortic regurgitation. CONCLUSIONS: In every patient in whom it was considered that a decision could be reached by echocardiography alone (more than 80% of patients) there was 100% agreement from the cardiac catheterization assessment group on the management decision. Therefore, in patients with significant mitral or aortic regurgitation where echocardiographic data is adequate, cardiac catheterization can be safely omitted from the investigative process for surgery. Where echocardiographic indices are conflicting, or significant coronary artery disease is suspected, cardiac catheterization is required.

[Does mitral insufficiency prevent spontaneous contrast phenomenon and formation of thrombi in the left atrium?]. / L'insuffisance mitrale prévient-elle la formation du phénomène de contraste spontané et la constitution des thrombus dans l'oreillette gauche?

The aim of this study was to assess the influence of mitral regurgitation on the prevalence of left atrial spontaneous echo contrast and thrombosis in 2,180 consecutive patients undergoing transthoracic and transoesophageal echocardiography. Two groups of patients were defined according to the absence (group I) or presence (group II) of grades 3 or 4 mitral regurgitation quantified by transoesophageal echocardiography. Group II was associated with a statistically significant lower frequency of spontaneous echo contrast (0.6 vs 11.2%; p < 0.0001), left atrial thrombosis (0.6 vs 4.2%; p < 0.03), ischaemic cerebrovascular accidents (1.2 vs 21%; p < 0.0001), transient ischaemic attacks (0 vs 12%; p < 0.0001) and systemic embolism (0 vs 4.6%; p < 0.01). Conversely, the prevalence of atrial fibrillation was higher in group II (28 vs 19%; p < 0.01) and there were more patients with left atrial dimensions > or = 5.5 cm (16 vs 6.7%; p < 0.0001). When mitral stenosis and valve prosthesis were excluded, there were no cases of spontaneous echo contrast (8.3 vs 0%; p < 0.001) or left atrial thrombosis (2.9 vs 0%; p < 0.05) in the group with grades 3 or 4 mitral regurgitation. The phenomenon of left atrial spontaneous echo contrast and/or thrombosis is rare in patients with grade 3 or 4 in native mitral valve regurgitation and explains the low incidence of systemic embolism in these cases.

Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.

The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbx1 oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.