Glial cell line-derived neurotrophic factor (GDNF) supports growth and survival of dopaminergic (DA) neurons. A replication-defective adenoviral (Ad) vector encoding human GDNF injected near the rat substantia nigra was found to protect DA neurons from the progressive degeneration induced by the neurotoxin 6-hydroxydopamine (6-OHDA) injected into the striatum. Ad GDNF gene therapy reduced loss of DA neurons approximately threefold 6 weeks after 6-OHDA lesion, as compared with no treatment or injection of Ad lacZ or Ad mGDNF (encoding a biologically inactive deletion mutant GDNF). These results suggest that Ad vector-mediated GDNF gene therapy may slow the DA neuronal cell loss in humans with Parkinson's disease.

PurposeThis study aims to study the effects of depression and demoralization on suicidal ideation and to determine the feasibility of the Distress Thermometer as a screening tool for patients with cancer who experience depression and demoralization, and thus to establish a model screening process for suicide prevention.MethodsPurposive sampling was used to invite inpatients and outpatients with lung cancer, leukemia, and lymphoma. Two hundred participants completed the questionnaire, which included the Distress Thermometer (DT), Patient Health Questionnaire-9 (PHQ-9), Demoralization Scale-Mandarin Version (DS-MV), and Beck Scale for Suicide Ideation. All data obtained were analyzed using SPSS 18.0 and SAS 9.3.ResultsTobit regression analysis showed that demoralization influenced suicidal ideation more than depression did (t = 2.84, p < 0.01). When PHQ-9 ≥ 10 and DS-MV ≥42 were used as criteria for the DT, receiver operating characteristic analysis revealed that the AUC values were 0.77–0.79, with optimal cutoff points for both of DT ≥5; sensitivity 76.9 and 80.6 %, respectively; and specificity of 73.9 and 72.2 %, respectively.ConclusionsDemoralization had more influence on suicidal ideation than depression did. Therefore, attention should be paid to highly demoralized patients with cancer or high demoralization comorbid with depression for the purposes of suicide evaluation and prevention. The DT scale (with a cutoff of ≥5 points) has discriminative ability as a screening tool for demoralization or depression and can also be used in clinical settings for the preliminary screening of patients with cancer and high suicide risk.

Transducing and distributing a vector throughout a tumor mass are presently insufficient for effective cancer gene therapy. To overcome these difficulties an adenoviral vector was designed that would replicate specifically in tumor cells. This tumor-specific replication-restricted adenoviral (TSRRA) vector was constructed by requiring that the essential E1A gene be expressed from a tumor-specific promoter, namely, the alpha-fetoprotein (AFP) gene promoter. This promoter was chosen since the AFP gene is highly expressed in 70-80% of patients with hepatocellular carcinoma (HCC) but not in normal adults. HCC is one of the major worldwide causes of cancer death. A vector was constructed (AvE1a04i) and demonstrated to replicate in human AFP-producing HCC cell lines. However, little replication was observed in seven other, non-AFP-producing human cell lines, as well as primary cultures of normal human lung epithelial and endothelial cells. In addition, AvE1a04i was shown to prevent tumor growth of an ex vivo-transduced AFP-expressing HCC cell line but not a non-AFP-expressing cell line. Finally, in situ administration of AvE1a04i into preestablished tumors resulted in a greater than 50% long-term survival rate. This novel TSRRA vector for HCC demonstrated both specificity and efficacy in vitro and in vivo.

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Introduction of the zta gene of Epstein-Barr virus into latently infected B cells leads to induction of the entire lytic cycle program of the virus. The Zta gene product is a sequence-specific DNA-binding protein of 35 kilodaltons that behaves as a specific transcriptional transactivator in transient cotransfection assays. All known Zta-responsive target promoters contain one or more members of a family of consensus-binding sites known as ZREs. On the basis of the presence of limited amino acid similarity within a basic carboxy-terminal domain, Zta has been proposed to be a highly divergent member of the c-Jun/c-Fos/GCN4 family of AP-1-binding proteins. We show here that in vitro-translated Zta and the Jun:Fos proteins have overlapping but distinct target DNA-binding specificies; both recognize canonical AP-1 sites, but only Zta recognizes ZRE sites and only Jun:Fos recognizes CRE sites. The relative binding affinity of Zta for oligonucleotides containing the 7-base-pair c-Fos AP-1 site TGAGTCA was twofold greater than that for the ZRE core motifs TGAGCAA, TG TGCAA, and TGAGTAA, but 10-fold greater than that for TGTGTCA, as measured by gel mobility retardation and competition DNA-binding assays. Cross-linking and cotranslational heterodimerization assays showed that like GCN4, Zta forms a stable homodimer in both its DNA-bound and unbound forms. Furthermore, we show that a potential coiled-coil helical domain adjacent to the basic domain of Zta can substitute for the leucine zipper of c-Fos to produce a DNA-binding protein that has a very stringent target DNA specificity and can only recognize symmetric 9-base-pair AP-1 sites (ATGAGTCAT). Therefore, despite the absence of the repeated heptad leucine zipper motifs, the Zta protein retains the characteristic features of a juxtaposed basic region and an exactly aligned coiled-coil alpha-helical dimerization domain of the bZIP class of transcriptional regulatory factors.

The IE2 region of the human cytomegalovirus (CMV) strain Towne major immediate-early (MIE) gene encodes a transcriptional transactivator that stimulates expression from a variety of heterologous target promoters but specifically down-regulates its own promoter. By immunofluorescence and Western immunoblot analysis with monospecific peptide antisera, we found that human CMV MIE exon 5 encodes four overlapping polypeptides, two present at immediate-early times (80 and 55 kDa) and two others detected only at late times after infection (55 and 40 kDa). However, only the 80-kDa version (579 amino acids), which is derived from the small upstream exons 2 and 3 fused to the intact exon 5 region, was functionally active in both transactivation and autoregulation as assessed by cotransfection experiments. These results confirm the corrected assignment of the coding capacity of the exon 5 region based on amino acid homology with the equivalent IE2 protein from simian CMV (Colburn). In transient DNA transfection assays, IE2 expression plasmids also produced a predominant full-length 80-kDa protein, which was localized in a distinctive reticular pattern in the nucleus. Two short basic nuclear localization signals in IE2 were identified by deletion analysis and by conversion of a test cytoplasmic herpes simplex virus protein into a form that localized in the nucleus after insertion of either of these two human CMV motifs. Functional assays with MIE region plasmids containing deletions or truncations in exon 5 revealed that both transactivation and autoregulation required several distinct domains within the COOH half of the IE2 protein, whereas a region between codons 99 and 194 could be discarded. Three segments at the NH2 end of the protein between codons 1 to 85, 86 to 98, and 195 to 290 were also essential for transactivation but played no role in autoregulation. Finally, in domain swap experiments, GAL4-fusion proteins containing either an NH2-terminal 51-amino-acid domain from exon 3 (codons 25 to 85) or the COOH-terminal 33-amino-acid domain from exon 5 (codons 544 to 579) identified two distinct activator domains from IE2, both of which have acidic characteristics.

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Objective-Inosine is a naturally occurring nucleoside degraded from adenosine. Recent studies have demonstrated that inosine has potent immunomodulatory and neuroprotective effects. In the present study, we further investigated the inhibitory effects of inosine on platelet activation in vitro and in vivo, as well as in attenuating middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Methods and Results-Inosine concentration-dependently (0.5 to 6.0 mmol/L) inhibited platelet aggregation stimulated by agonists. Inosine (1.5 and 3.0 mmol/L) inhibited phosphoinositide breakdown, [Ca ϩ2 ]i, and TxA 2 formation in human platelets stimulated by collagen (1 g/mL). In addition, inosine (1.5 and 3.0 mmol/L) markedly increased levels of cyclic guanylate monophosphate (GMP) and cyclic GMP-induced vasodilator-stimulated phosphoprotein Ser 157 phosphorylation. Rapid phosphorylation of a platelet protein of molecular weight 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 g/mL). This phosphorylation was markedly inhibited by inosine (3.0 mmol/L). Inosine (1.5 and 3.0 mmol/L) markedly reduced hydroxyl radical in collagen (1 g/mL)-activated platelets. In in vivo studies, inosine (400 mg/kg) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules of mice, and administration of 2 doses (100 mg/kg) or a single dose (150 mg/kg) of inosine significantly attenuated MCAO-induced focal cerebral ischemia in rats. Conclusions-Platelet aggregation contributes significantly to MCAO-induced focal cerebral ischemia. The most important findings of this study suggest that inosine markedly inhibited platelet activation in vitro and in vivo, as well as cerebral ischemia. Thus, inosine treatment may represent a novel approach to lowering the risk of or improving function in thromboembolic-related disorders and ischemia-reperfusion brain injury. (Arterioscler Thromb Vasc Biol.