Anxiety, helplessness/hopelessness and 'desire for hastened death' in Korean cancer patients.

Despite a relatively high rate of suicide associated with cancer, this issue has not been explored in Korean patients. This study investigates the prevalence and factors related to 'the desire for hastened death' (DHD) in Korean cancer patients. A cross-sectional survey using standardised measures, including the Schedule of Attitudes toward Hastened Death and the Hospital Anxiety and Depression Scale, was performed with 131 patients with different types of cancer. 13.7% of the participants experienced moderate DHD (Schedule of Attitudes toward Hastened Death scores 5-9) and 1.7% experienced high DHD (≥10). Socio-demographic and disease-associated factors of the DHD included age, overall health and shortness of breath. The majority of psychosocial variables such as sadness, distress, 'helplessness/hopelessness' and 'anxious preoccupation' had a moderate association with DHD. Patients with a clinically significant level of anxiety or depression reported higher levels of DHD. Other significant correlates included 'meaning/peace', a sense of burdening family, dignity impairment and suicidal thoughts after diagnosis. Helplessness/hopelessness and anxiety were the strongest predictors of DHD in multivariate analysis. In view of significant role of helplessness/hopelessness and anxiety in the DHD of cancer patients, careful monitoring and management of these factors should be an integral part of cancer care to reduce the occurrence of DHD.

Depression and health-related quality of life in maintenance hemodialysis patients.

BACKGROUND: This study was designed to determine the prevalence of depression among hemodialysis (HD) patients from urban hospitals in Korea, to illustrate demographic factors and biomarkers associated with depression and health-related quality of life (HRQOL), and to demonstrate association between depression and HRQOL. PATIENTS AND METHODS: For this multicenter, cross-sectional study, 160 HD patients from 3 university teaching hospitals and 3 local dialysis units in Korea were enrolled. Korean Beck's depression inventory and Korean version of Kidney Disease Quality of Life short form, version 1.3 (KDQOL-SFTM 1.3) were used to evaluate depression and quality of life, respectively. RESULTS: Depression was found in 51 out of 160 (31.9%) patients. Old age (> 60 years old), low hemoglobin level (< 10 g/dl), and low economic status were associated with depression, and old age (OR 6.138, p = 0.001) was the most important risk factor among them. Old age, female gender, presence of diabetes mellitus, high comorbidity index score (modified Charlson comorbidity index > or = 6), hypoalbuminemia (< 4.0 g/dl), and high CRP (> 0.5 mg/dl) were common factors associated with decreased HRQOL. Depression and HRQOL showed inverse linear relationship. CONCLUSIONS: Moderate to severe depression was common in maintenance HD patients in Korea. Among factors associated with depression and decreased HRQOL, some characteristics are potentially modifiable by social and medical intervention. Further prospective studies are warranted to see whether depression and HRQOL can be improved by modifying these factors.

Hostile communication of measles virus with host innate immunity and dendritic cells.

Following measles virus (MV) infection, host innate immune responses promptly operate to purge the virus. Detection of alerting measles viral components or replication intermediates by pattern-recognizing host machinery of Toll-like receptors and RNA helicases triggers signaling to synthesize array of anti-viral and immunoregulatory molecules, including type I interferon (IFN). Diverse subtypes of dendritic cells (DCs) play pivotal roles in both host innate immunity on the primary MV-infected site and initiating adaptive immune responses on secondary lymphoid tissues. Responding to the predictable host immune responses, MV appears to have devised multiple strategies to evade, suppress, or even utilize host innate immunity and DC responses. This review focuses on versatile actions of MV-induced type I IFNs causing beneficial or deleterious influence on host immunity and the interplay between MV and heterogeneous DCs at distinct locations.

Depression and the vision-related quality of life in patients with retinitis pigmentosa.

AIMS: To assess the relationship between depression and the vision-related quality of life in patients with retinitis pigmentosa (RP). METHODS: The study included 144 patients diagnosed as having RP. The mean age of the patients was 38.5 (SD 13.3) years, and 42% of the subjects were women. They answered the National Eye Institute Visual Function Questionnaire (NEI-VFQ) to assess the vision-related quality of life and the Beck Depression Inventory (BDI) to assess depressive symptoms. Patients were classified into groups with and without depression according to the BDI score. The NEI-VFQ composite and subscale scores were compared between groups. The correlations between the BDI and the NEI-VFQ, weighted visual acuity (WVA) and functional vision score (FVS) were investigated. RESULTS: The depressed group had significantly less subjective visual function compared with the non-depressed group. A negative correlation was observed between the BDI and the NEI-VFQ scores, while no correlation was found between the BDI score and WVA or FVS. CONCLUSION: The RP patients with depression had poorer vision-related functions compared with those patients without depression, which cannot be explained by the visual acuity. Interventions to diagnose and treat depression are necessary to enhance the overall quality of life in RP patients.

Type I interferon during viral infections: multiple triggers for a multifunctional mediator.

Type I interferons (IFN-I) orchestrate numerous biological and cellular processes and are essential elements during host antiviral defense. After recognition of highly conserved virus signatures, a complex network of signaling events is rapidly initiated and leads to IFN-I synthesis. These cytokines directly induce a strong antiviral state and exert several immune-regulatory actions aimed at preventing virus spread. On the other hand, viruses evolved to evade or subvert the IFN-I system for their own benefit. In the present article, we review selective aspects of IFN-I induction and functions during several viral infections and discuss the beneficial and detrimental roles of IFN-I illustrated during lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse.

Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes.

AIMS: To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. METHODS AND RESULTS: Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45 degrees C), NaCl (5.5%), oxidative stress (15 mmol(-1) H2O2), acidic pH (5.5) and ethanol (5%) for 3 h (short-term stress) or for 5 days (long-term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12-16% reductions in reaction for anti-E. coli O157:H7 and 20-48% reductions for anti-Salmonella polyclonal antibodies during short-term stress, whereas the most stresses exhibited enhanced reaction (44-100% increase) with the anti-L. monocytogenes polyclonal antibody. During long-term stress exposure to combined stress conditions of pH 5.5, 3.5% NaCl at 12 degrees C or at 4 degrees C, antibody reactions to the three pathogens were highly variable with the combined stress at 4 degrees C showing the most reductions (8-40%). Likewise, there were about 18-59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short- and long-term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. CONCLUSIONS: Overall, the antibody reactions were significantly reduced in pathogens exposed to both short- and long-term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.

Polypyrimidine tract-binding protein inhibits translation of bip mRNA.

Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. No trans-acting factor possibly involved in this process has as of yet been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. Here, we report that PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibited Bip IRES-dependent translation. On the other hand, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs.

Expression and purification of an active, full-length hepatitis C viral NS4A.

The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.

Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3.

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.

Protein-protein interaction among hnRNPs shuttling between nucleus and cytoplasm.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in several RNA-related biological processes such as transcription, pre-mRNA processing, mature mRNA transport to the cytoplasm, and translation. About 20 major hnRNPs from A1 to U are known. Among them, hnRNP A, D, E, I, and K are known to shuttle between the nucleus and the cytoplasm. hnRNP E2 has been seen to stabilize alpha-globin mRNA and to enhance polioviral mRNA translation. hnRNP K modulates transcription and translation of some mRNAs. hnRNP I and its homologue hnRNP L have been suggested to enhance translation of some IRES-dependent mRNAs. In order to better understand the molecular mechanisms of the biological functions of hnRNPs, we investigated protein-protein interactions of six hnRNPs (hnRNP A1, C1, E2, I, K, and L) using the yeast two-hybrid system and in vitro co-precipitation assays. All of the hnRNPs tested exerted homomeric interactions, and hnRNP E2, I, K, and L interacted with each other. In the case of hnRNP E2 and hnRNP K, the N-terminal half of the proteins containing two KH (K homologous) domains were required for protein-protein interaction, and the second quarter of hnRNP I and hnRNP L containing RRM2 (RNA recognition motif 2) was essential for protein-protein interaction. hnRNP A1 and C1 did not form complexes with other hnRNPs in our assay systems. This suggests that the hnRNPs could fall into two groups: one group, including hnRNP A1 and C1, involved in hnRNP core complex formation and another group, including hnRNP E2, I, K, and L, involved in a variety of RNA-related biological processes. Different combinations of the proteins of the second group may facilitate different biological processes in conjunction with other factors.

Use of PCR to screen for promoter elements in genomic DNA library clones.

We report a modified PCR strategy to screen for promoter elements of genes of interest that is based upon consecutive rounds of PCR and appropriate subcloning. Following preliminary identification and sequencing of intron 1 by standardized PCR, the application of a suited cDNA/intron primer combination renders a succeeding PCR-mediated screening of cosmid or P1-derived artificial chromosome (PAC) libraries possible, thus identifying genomic clones comprising the searched promoter elements. We tested our approach in comparison with a commercially available promoter finder kit by searching the promoter elements of the CENP-C gene from the human and mouse genomes. Applying the kit system, we amplified the anticipated promoter from mouse, but failed in isolating human promoter elements. Our approach made use of a 5'-UTR/intron 1 primer combination in the second round of PCR, enabling the identification of positive clones from genomic DNA within a human PAC library possible. Subcloning and final PCR amplification revealed the successful isolation of the human promoter. Therefore, we conclude that our approach might represent a helpful alternative to identify promoter elements, especially when prior art genome walking, STS-based strategies or anchored PCR failed.

Heterogeneous nuclear ribonucleoprotein L interacts with the 3' border of the internal ribosomal entry site of hepatitis C virus.

Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5' nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3' border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translation of HCV mRNA through the IRES element.

Determination of functional domains in polypyrimidine-tract-binding protein.

Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169-293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329-530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).

Polypyrimidine tract-binding protein interacts with HnRNP L.

Polypyrimidine tract-binding protein (PTB) is involved in pre-mRNA splicing and internal ribosomal entry site (IRES)-dependent translation. In order to identify cellular protein(s) interacting with PTB, we performed a yeast two-hybrid screening. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as a PTB-binding protein. The interaction between PTB and hnRNP L was confirmed in an in vitro binding assay. Both PTB and hnRNP L were found to localize in the nucleoplasm, excepting the nucleoli, in HeLa cells by the green fluorescent protein (GFP)-fused protein detection method. The N-terminal half of PTB (aa 1-329) and most of hnRNP L (aa 141-558) is required for the interaction between PTB and hnRNP L.

Generation of a novel poliovirus with a requirement of hepatitis C virus protease NS3 activity.

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis. One of the difficulties in developing anti-HCV drugs is the lack of an efficient HCV cultivation system. We have generated an artificial surrogate virus suitable for testing the antiviral effects of drugs affecting HCV protease NS3, an enzyme believed to be essential for HCV proliferation. The surrogate virus genome is composed of most of the poliovirus genome and HCV protease NS3 and an NS3-specific cleavage site. The activity of HCV protease NS3 is required for proliferation of this chimeric virus. The antiviral efficacy of HCV protease inhibitors can, therefore, be evaluated by examining the effects of the drugs on the surrogate virus proliferation.

NS3-4A of hepatitis C virus is a chymotrypsin-like protease.

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.

Identification of the protease domain in NS3 of hepatitis C virus.

NS3 of hepatitis C virus (HCV) is a serine protease that carries out the proteolytic processing of the nonstructural proteins of the HCV polyprotein. Deletion analysis of the N terminus of NS2,3,4 fusion protein revealed that the N-terminal boundary of the active protease resides between amino acids 1050 and 1083. The processing patterns of internal deletion mutants of NS2,3,4 indicated that the C terminus of the enzymically active protease resides between amino acids 1115 and 1218. The N- and C-terminal boundaries of the protease were also confirmed by determining the trans-cleavage activity of internally deleted NS3,4. NS3 protease activity was inhibited by Cu2+ but was slightly enhanced by Zn2+. This report provides a possible approach for development of antiviral agents based on protease inhibitors.

Nucleotide sequence of the pectate lyase gene from alkali-tolerant Bacillus sp. YA-14.

The nucleotide sequence of the pectate lyase gene (pe lK) from alkali-tolerant Bacillus sp. was identified and analyzed. A 1,260-base pair open reading frame for the pe lK gene was observed and encoded for a protein of 420 amino acids. The signal peptide was composed of 21 amino acid residues. In the deduced primary structure of this enzyme, the three conserved regions of several pectate lyases were found and showed high homologies.

An experimental vaccine cocktail for Plasmodium falciparum malaria.

Surface proteins from several different life-cycle stages of the malaria parasite Plasmodium falciparum were expressed at high levels in the yeast Saccharomyces cerevisiae. Purified proteins, both individually and in cocktails, were used to immunize mice and goats in conjunction with either Freund's adjuvant or a muramyl tripeptide-based adjuvant. Immune responses were measured by enzyme-linked immunosorbent assays and by the ability of antisera to inhibit (1) the invasion of hepatocytes by live sporozoites, (2) in vitro invasion of human erythrocytes by live merozoites, and (3) the development of oocytes in the mosquito vector. These results suggest that cocktails of different stage-specific antigens can provide the components necessary to block the development of the malaria parasite at multiple stages of its life cycle.

Muramyl peptide adjuvants for Plasmodium falciparum and Plasmodium vivax circumsporozoite vaccines in rodent model systems.

Circumsporozoite proteins from the malaria parasites Plasmodium falciparum and Plasmodium vivax were expressed at high levels in the yeast Saccharomyces cerevisiae. Recombinant proteins varied both in length and in number of the natural amino acid repeat motifs. The proteins were purified and used to immunize mice, guinea pigs, and rabbits. Novel muramyl peptide adjuvants were used that increased the immune response as measured by ELISA assays, indirect immunofluorescence of fixed sporozoites, and the invasion of cultured liver cells by live sporozoites. These results suggest that an improved humoral response to recombinant circumsporozoite vaccines might be achieved by varying the design of the recombinant protein and by the use of novel adjuvant systems.