Mutations in the core and NS5A region of hepatitis C virus genotype 1b and correlation with response to pegylated-interferon-alpha 2b and ribavirin combination therapy.

Mutations in two regions of hepatitis C virus (HCV) have been implicated in influencing response to interferon (IFN) therapy. Substitutions in the NS5A region of HCV have been associated with response to IFN therapy, and this region has been known as the IFN sensitivity-determining region (ISDR). The mutations in the core region of HCV have also been reported to predict IFN response. The aim of this study was to investigate whether amino acid substitutions in the core region and ISDR among patients with HCV genotype 1b affect the response to IFN therapy. A total of 213 patients who completed IFN treatment were randomly selected. All patients received pegylated-IFN-alpha 2b once each week, plus oral ribavirin daily for 48 weeks. Of the 213 patients, 117 (54.9%) showed early virologic response (EVR), with HCV-negativity, at 12 weeks. Factors related to EVR on multivariate analysis were non-Gln70 and Leu91 in the core region, and ISDR mutant-type. One hundred and two (47.9%) showed a sustained virologic response (SVR). SVR occurred more frequently in patients without Gln70 (55.4%) than in those with Gln70 (21.3%) (P < 0.0001). SVR was achieved in 43.6% of patients with wild-type ISDR and 62.5% of patients with mutant-type (P = 0.0227). Of the 34 patients who simultaneously had non-Gln70 and mutant-type ISDR, 26 (76.5%) achieved SVR. Factors related to SVR on multivariate analysis were non-Gln70 and ISDR mutant-type. In conclusion, amino acid substitutions in the core region and ISDR were useful for predicting the response to IFN in patients with HCV genotype 1b.

Upregulation of BiP and CHOP by the unfolded-protein response is independent of presenilin expression.

Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.

A switch from life to death in endoplasmic reticulum stressed ß-cells.

ß-Cell death is an important pathogenic component of both type 1 and type 2 diabetes. Recent findings indicate that cell signalling pathways emanating from the endoplasmic reticulum (ER) play an important role in the regulation of ß-cell death during the progression of diabetes. Homeostasis within the ER must be maintained to produce properly folded secretory proteins, such as insulin, in response to the body's need for them. However, the sensitive protein-folding environment in the ER can be perturbed by genetic and environmental factors leading to ER stress. To counteract ER stress, ß-cells activate cell signalling pathways termed the unfolded protein response (UPR). The UPR functions as a binary switch between life and death, regulating both survival and death effectors. The outcome of this switch depends on the nature of the ER stress condition, the regulation of UPR activation and the expression and activation of survival and death components. This review discusses the mechanisms and the components in this switch and highlights the roles of this UPR's balancing act between life and death in ß-cells.

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.

Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5'UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5'UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5' leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Neuroleptic malignant syndrome with prolonged catatonia in a dopa-responsive dystonia patient.

The authors describe a patient with dopa-responsive dystonia who developed neuroleptic malignant syndrome with prolonged catatonia following treatment with neuroleptic agents. Use of these agents probably expanded the patient's neuronal dysfunction beyond the nigrostriatal system to involve multiple dopaminergic systems. Electroconvulsive treatment alleviated the prolonged catatonia.

Acute myeloid leukemia possessing jumping translocation is related to highly elevated levels of EAT/mcl-1, a Bcl-2 related gene with anti-apoptotic functions.

Jumping translocations (JTs) are unbalanced chromosomal translocations in which an identical chromosomal region is translocated to the telomeric region of different chromosomes. JTs are rare in hematological malignancies where they are second translocations and may be an indicator of poor prognosis. We report a case of acute myeloid leukemia with t(16;21) and a JT in which the long arm of chromosome 1 distal to q21 is translocated to the terminal region of chromosome 10. The leukemic cells exhibit high expression of EAT/mcl1, an anti-apoptotic Bcl-2 related gene. Since EAT/mcl1 is mapped to 1q21 near the breakpoint in the JTs, high level expression of EAT/mcl1 may be associated with the poor prognosis of leukemia with JTs.

Evaluation of intraaneurysmal blood velocity by time-density curve analysis and digital subtraction angiography.

PURPOSE: Our purpose was to evaluate intraaneurysmal blood velocity by using time-density curve analysis and digital subtraction angiography. METHODS: In 31 aneurysms, aneurysmal blood velocity was examined with digital subtraction angiography to determine mean transit time (MTF), peak density time (time to peak opacification) (PDT), and time to half-peak opacification (T1/2). Thirty frames per second were acquired, and the time-density curve was calculated. Regions of interest were drawn on the proximal parent artery, on the distal parent artery, and on the aneurysm itself. RESULTS: There was no significant difference in MTT of blood velocity in the proximal site on the parent artery, in the distal site on the parent artery, and in the aneurysm. Similarly, there was no significant difference in PDT in the parent artery, in the distal site on the parent artery, and in the aneurysm; nor was there a significant difference in T1/2 in the parent artery, in the distal site on the parent artery, and in the aneurysm; that is, intraaneurysmal blood velocity was similar to that in the parent artery. PDT and T1/2 of small aneurysms were faster than that of large aneurysms; that is, blood velocity of small aneurysms was faster than that of large aneurysms. CONCLUSION: Intraaneurysmal blood velocity in small aneurysms is similar to that in the parent artery; consequently, the central stream probably reaches the aneurysmal wall. Intraaneurysmal blood velocity in large aneurysms appears to be somewhat slower than that in small aneurysms.

Prophylactic endoscopic ligation of high-risk oesophageal varices in a cirrhotic patient with severe haemophilia A.

One of the main complications in cirrhosis is haemorrhage from oesophageal varices. It is serious and often fatal, especially in cirrhotic patients with haemophilia. We describe the use of endoscopic variceal ligation (EVL) for prophylaxis of oesophageal variceal bleeding in a high-risk patient, a 40-year-old Japanese man, with severe haemophilia A and liver cirrhosis caused by hepatitis C virus. He had large, coil-shaped varices with a red colour sign, predicting the likelihood of haemorrhage. Administration of omeprazole and factor VIII concentrate achieved rapid healing of the post-EVL ulcers and prevented bleeding from them. Four EVL sessions eradicated oesophageal varices completely, and he has had no recurrence of varices for 2 years, indicating that the procedure was of considerable benefit.

Serum levels of soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 in liver disease, and their changes by treatment with interferon.

Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured by an enzyme-linked immunosorbent assay in patients with chronic hepatitis (n = 57), liver cirrhosis (n = 19) and hepatocellular carcinoma (n = 33). Serum levels of sICAM-1 and sVCAM-1 were significantly higher in liver disease than those in controls (P < 0.0001 and P < 0.0005, respectively). A total of 22 patients with chronic hepatitis C were treated with interferon. Pretreatment levels of sICAM-1 and sVCAM-1 were not significantly different between complete responders and non-responders. In complete responders, serum sICAM-1 and sVCAM-1 levels 1 year after interferon treatment significantly decreased compared to the pretreatment levels (P < 0.005 and P < 0.05, respectively). Post-treatment levels of sICAM-1 and sVCAM-1 in complete responders were also significantly lower than those in non-responders (P < 0.005 and P < 0.05, respectively). This suggests that monitoring soluble adhesion molecules might be useful in the follow-up of patients with liver disease.

[Early detection of primary liver cancer--diagnosis of small liver cancer by needle aspiration biopsy].

Small liver cancer is defined as a solitary hepatocellular carcinoma (HCC) with a diameter less than 2cm. To detect liver cancer as early as possible, patients with liver cirrhosis are screened by ultrasound scanning. Pathological diagnosis in needle aspiration biopsy materials is needed because of low positivity of imaging other than ultrasound scanning. Pathological features are different from those of classical hepatocellular carcinoma. Most of the small HCCs are characterized by the following features: (1) increased cellularity, (2) increased nucleus/cytoplasm ratio, (3) irregular thin trabecular pattern, (4) pseudoglandular or acinar structures, (5) increased staining affinity (eosinophilic/basophilic), (6) frequent fatty change, and (7) residue of the portal tract. Capsules of HCCs, 1-1.5 cm in diameter, are formed. Before the formation of capsules, cancerous cells show a replacing growth pattern. Two cases of small HCC are presented, and these characteristic features are explained.

[Value of MR cholangiography in the diagnosis of pancreatic diseases compared with endoscopic retrograde cholangiopancreatography].

PURPOSE: To compare magnetic resonance cholangiography (MRCP) with endoscopic retrograde cholangiography (ERCP) in the diagnosis of pancreatic diseases. METHODS: The authors retrospectively reviewed MRCP and ERCP images obtained in 56 patients with pancreatic diseases and 192 controls. Detectability of the pancreatic duct and its branches and diagnostic usefullness were evaluated. RESULTS: MRCP was successful in almost all the patients without ascitic fluid or ileus. Findings of the main pancreatic ducts (i.e., dilation, stenosis and obstruction) were similarly demonstrated by ERCP and MRCP. The degree of ductal narrowing tends to be overestimated with MRCP. CONCLUSION: MRCP appears to be less invasive than ERCP. MRCP could provide sufficient information and play a complementary role in the diagnosis of pancreatic diseases.

Activation of OASIS family, ER stress transducers, is dependent on its stabilization.

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1(-/-) cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1(-/-) cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1.

Endoplasmic reticulum (ER) stress-mediated cell death has an important role in the pathogenesis of chronic diseases, including diabetes and neurodegeneration. Although proapoptotic programs activated by ER stress have been extensively studied, identification and characterization of antiapoptotic programs that counteract ER stress are currently incomplete. Through the gene expression profiling of beta-cells lacking Wolfram syndrome 1 gene (WFS1), a causative gene for Wolfram syndrome, we discovered a novel antiapoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here, we study the regulation of AATF, identify its target genes, and determine the basis for its antiapoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK-eIF2alpha pathway and transcriptionally activates the v-akt murine thymoma viral oncogene homolog 1 (AKT1) gene through signal transducer and activator of transcription 3 (Stat3), which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered a positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1 deficiency or AATF deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel antiapoptotic program relevant to the treatment of diseases caused by ER stress-mediated cell death.