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The aim is to explore the impact of violence and psychological resilience on psychiatric nurses as second victims.Workplace violence is a public health concern, seriously influencing medical staff's physical and mental health. However, few pieces of research have concentrated on psychiatric nurses as second victims.The socio-demographic data, violence-related data, psychological resilience scale, the Chinese version of the Second Victim Experience and Support Tool were applied to conduct a cross-sectional survey on nurses from psychiatric hospitals. The multiple linear regression model identified significant variables associated with violence-related injury and resilience.A total of 683 nurses completed the survey, of whom 88.3% were women. The average scores of the second victims' experience and support, support and distress were 3.45 ± 0.43, 3.71 ± 0.57 and 3.19 ± 0.67, respectively. Physical violence, psychological violence, psychological influence and nursing work environment were positively correlated with the experience and support of the second victims, and innocuous violence was a negative factor, which explains 20.6% of the variation. Moreover, physical injury, nursing work environment, resilience restructuring, physical violence, psychological violence, psychological impact and disagreement about the existence of violence explained 14.8% of the distress. The innocuous violence, psychological violence and nursing work environment explained 46.2% of the support.Our findings suggest that nurses who are second victims after the violence mainly suffer from severe psychological distress and receive the least support for the same.The study provides clues to help nursing managers' emphasis on the distress and support of second victims and provides targeted intervention based on the relevant factors and these results. The article is a cross-sectional study of psychiatric nurses, which has been approved by the ethics committee of the hospital before the survey. All the nurses who participated in the survey have been informed and agreed.

Rabies kills around 60 000 people each year. ChAdOx2 RabG, a simian adenovirus-vectored rabies vaccine candidate, might have potential to provide low-cost single-dose pre-exposure rabies prophylaxis. This first-in-human study aimed to evaluate its safety and immunogenicity in healthy adults.We did a single-centre phase 1 study of ChAdOx2 RabG, administered as a single intramuscular dose, with non-randomised open-label dose escalation at the Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK. Healthy adults were sequentially allocated to groups receiving low (5 × 10Between Jan 2 and Oct 28, 2020, 12 adults received low (n=3), middle (n=3), and high doses (n=6) of ChAdOx2 RabG. Participants reported predominantly mild-to-moderate reactogenicity. There were no serious adverse events. Virus neutralising antibody concentrations exceeded the recognised correlate of protection (0·5 IU/mL) in three middle-dose recipients and six high-dose recipients within 56 days of vaccination (median 18·0 IU/mL). The median peak virus neutralising antibody concentrations within 56 days were 0·7 IU/mL (range 0·0-54·0 IU/mL) for the low-dose group, 18·0 IU/mL (0·7-18·0 IU/mL) for the middle-dose group, and 18·0 IU/mL (6·0-486·0 IU/mL) for the high-dose group. Nine participants returned for the additional follow-up after 1 year. Of these nine participants, virus neutralising antibody titres of more than 0·5 IU/mL were maintained in six of seven who had received middle-dose or high-dose ChAdOx2 RabG. Within 7 days of administration of the first dose of a licensed rabies vaccine, nine participants had virus neutralising antibody titres of more than 0·5 IU/mL.In this study, ChAdOx2 RabG showed an acceptable safety and tolerability profile and encouraging immunogenicity, supporting further clinical evaluation.UK Medical Research Council and Engineering and Physical Sciences Research Council.

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BACKGROUND Complications are the most important outcome determinants for acute pancreatitis (AP). We designed this single-center retrospective study to evaluate the clinical findings (complications, disease severity, and outcomes) of 218 patients with AP and to identify variables associated with ascites. MATERIAL AND METHODS We extracted clinical data from consecutive patients with AP and divided them into 2 groups based on presence or absence of ascites. We compared disease severity, complications, and outcomes between groups. RESULTS We analyzed data from 218 patients with AP (43 with ascites and 175 without it). The patients with ascites had a more severe disease (higher incidence of pancreatic inflammation [90.70% vs 68.57%; P=0.003], higher modified computed tomography severity index score [2.00 (0.00-2.00) vs 4.00 (4.00-6.00); P

Six new polyketides, alternaritins A–D [(±)-1–4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1–9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

BACKGROUND Renal dysfunction is a leading cause of death in patients with acute pancreatitis (AP) and often occurs later than respiratory complications. Whether respiratory complications can predict renal impairment remains unclear. The aim of this study was to investigate the association between pleural effusion and renal dysfunction in AP. MATERIAL AND METHODS Medical records were reviewed from individuals who were hospitalized with AP from January 1, 2015 to December 31, 2019. The patients were divided into 2 groups, based on the presence or absence of pleural effusion on admission. Disease severity, renal function parameters, and outcomes were compared between the 2 groups. RESULTS A total of 222 patients were enrolled, 25 of whom had pleural effusion on admission and 197 who did not. Patients with AP who had pleural effusion had more serious illness (higher incidences of pancreatic inflammation, pancreatic fluid collection, and moderate-to-severe AP; worse Bedside Index for Severity in Acute Pancreatitis score; and a higher modified computed tomography severity index [all P

Carpesium humile Winkl is an endemic Chinese species and no previous phytochemical studies have been reported for this species. Two new germacranolides (1 and 2) and a new phytane diterpene (5), together with five known compounds (two sesquiterpenoids and three diterpenoids), were isolated from the aerial parts of C. humile. Their structures were elucidated on the basis of extensive spectroscopic analysis. The conformations and absolute configurations of 1 and 2 were established by combinative analysis of NMR, CD exciton chirality, and X-ray crystallography data. Four germacranolides (1–4) showed strong cytotoxic activities, with broad spectrum activities against six human cancer (HepG2, HeLa, HL60, SGC7901, Lewis, and MDA231) cell lines in vitro using MTT assay, with IC50 values from 3.09 to 7.71 μg/mL. Diterpenes (5, 6, and 8) also displayed good cytotoxic activities for selected cancer cell lines, with IC50 values in the range 5.46–8.08 μg/mL.

Extracts of the aerial parts of Scorzonera divaricata afforded sulfoscorzonin D (1) and sulfoscorzonin E (2), two novel pyrrolidine inner salt alkaloids with a sulfated guaiane sesquiterpene lactone nucleus, along with 22 known compounds. Especially, sulfoscorzonin D containing a unusual monoterpene moiety is very rare. The structures of new compounds were established using spectroscopic analysis including one- and two-dimensional NMR and HRESIMS. The cytotoxicities of compounds 1–4 and 10 against three tumor cell lines (K562, Hela, and HepG2) were evaluated using the MTT assay. Compounds 2 and 10 exhibited moderate cytotoxic activity. The biological properties of 1–3, 5–8, 10–14, and 16–24, were screened against nine different gram-positive and gram-negative bacteria. Compounds 1, 5–8, 10, and 18, showed potent antibacterial activities. Chemical compounds studied in this article Glucozaluzanin C (PubChem CID: 442320); 1β,4α-dihydroxy-5α,6β,7α,11βH-eudermn-12; 6-olide (CID: 11119093); oleanolic acid (CID: 10494); lup-20(29)-ene-3β,28-diol (CID: 72326); (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (CID: 5469431); ergosta-3β,5α, 6β-trialcohol (CID: 44558918); stigma-5-en-3-O-β-glucoside (CID: 5742590); vomifoliol (CID: 12444927); trans-caffeic acid (CID: 689043); trans-p-hydroxy coumaric acid (CID: 637542); 4-hydroxy-3-methoxyphenyl ferulate (CID: 11500646); 7,3′,4′-trihydroxyflavonol (CID: 5281614); tricin (ID: 5281702); luteolin (CID: 5280445); diosmetin (CID: 5281612); 5,7-dihydroxy-8-methoxyflavone (CID: 5281703); 5,7-dihydroxy-6-methoxyflavone (CID: 5320315); methyl-3,4-dihydroxy benzoate (CID: 287064); m-hydroxy benzoic acid (CID: 7420); 7-hydroxy-coumarin (CID: 5281426); and scopoletin (CID: 5280460).

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8+ T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8+ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8+ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.

CD8+ T cell-numbers rapidly expand and then contract after exposure to their cognate antigen. Here we show that the sustained frequencies of transgene product-specific CD8+ T cells elicited by replication-defective adenovirus vectors are linked to persistence of low levels of transcriptionally active adenovirus vector genomes at the site of inoculation, in liver, and lymphatic tissues. Continuously produced small amounts of antigen maintain fully active effector CD8+ T cells, while also allowing for their differentiation into central memory cells. The long-term persistence of adenoviral vectors may be highly advantageous for their use as vaccines against pathogens for which T-cell–mediated protection requires both fully activated T cells for immediate control of virus-infected cells and central memory CD8+ T cells that, because of their higher proliferative capacity, may be suited best to eliminate cells infected by pathogens that escaped the initial wave of effector T cells.

Two new isocoumarins (1 and 2), a new alkaloid (3), and a known N-acetyldopamine dimer (4) were isolated from the ethyl acetate extract of Chinese insect medicine Eupolyphaga sinensis. Their structures were elucidated on the basis of detailed spectroscopic investigations, such as 1D- and 2D NMR spectroscopy, as well as by means of HR-MS. The structure of 1 was firmly confirmed by X-ray crystallography, and the absolute configuration was revealed by experimental and computational optical rotation analyses. Cytotoxicities of 1-4 were measured in vitro against 10 selected cancer cell lines.

A new oxazole (1) was obtained from Chinese insect medicine Aspongopus chinensis, along with three known N-acetyldopamine derivatives (2-4). Their structures were determined on the basis of NMR and ESI-MS analyses. The possible biosynthetic pathways of the isolated compounds are discussed. Cytotoxicities of those compounds against 10 selected cancer cells were measured in vitro.

A new D:B-friedoolean-type triterpene, 3β-acetoxyglutin-5(10)-en-6-oxo (1), together with seventeen known compounds (2-18) was isolated from the roots of Scorzonera austriaca. Their structures were elucidated mainly by NMR and HR-ESI-MS, as well as on comparison with the reported data. Cytotoxicities of compounds 2, 4, 6, 10-14 and 16 against selected cancer cells of human promyelocytic leukemia (HL-60) and human hepatoma (BEL-7404) were measured in vitro.

Azonazine, a unique hexacyclic dipeptide, was isolated from a Hawaiian marine sediment-derived fungus eventually identified as Aspergillus insulicola. Its absolute configuration, 2R,10R,11S,19R, was established using NMR, HRESIMS, and CD data plus insights derived from molecular models. A possible route for its biogenesis is proposed, and biological properties were explored against cancer cell lines and in an NFκB inhibition assay.

Hepatic adeno-associated virus (AAV)-serotype 2-mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid-specific CD8(+) T cells.

A total of 27 natural products were isolated from Saussurea cauloptera, including two new eudesmane-type sesquiterpenoids, the gerin derivatives 2 and 3, and one new ent-labdane diterpenoid, compound 9. The known compounds included six sesquiterpenoids, eleven triterpenoids, six sterols, and one lignan. Their structures were elucidated on the basis of extensive spectroscopic and mass-spectrometric analyses, as well as by X-ray crystallography in the case of gerin (1). The structurally related compounds 1-4 were found to exhibit strong inhibitory activities against human gastric carcinoma (SGC-7901) cells.

Two new, highly oxygenated ursane-type triterpenoids with an epidioxy function, compounds 1 and 2, as well as a new stanside-derived iridoid, compound 10, were isolated from the EtOH extract of Gentiana aristata (whole plant), together with 14 known constituents. The structures of the new compounds were established on the basis of detailed spectroscopic investigations, such as 1D- and 2D-NMR spectroscopy, as well as by means of HR-MS. Compounds 3 and 5-9 were screened in vitro for their cytotoxicities against human promyelocytic leukemia (HL-60), human ovarian (HO-8910), and human lung epithelia (A-549) cancer cells.

Two novel ent-kaurane diterpenoids, taihangjaponicain A (1), and taihangjaponicain B ( 2), and nine known diterpenoids, epinodosin (3), oridonin (4), epinodosinol (5), lasiokaurin ( 6), 1alpha- O-beta- D-glucopyranosylenmenol (7), lasiodonin (8), rabdosichuanin D ( 9), shikokianin (10) and rabdoternin A (11) were isolated from I. japonica leaves. The structures of the two new compounds were elucidated using 1-D and 2-D NMR spectroscopy. Compounds 1 and 3 - 11 were tested against HL-60, HO-8910 and A-549 human tumor cells. Compounds 4, 6 and 10 showed significant cytotoxicity against HL-60 cells with IC (50) values of 4.6, 2.0 and 3.4 microM, respectively, and against A-549 cells with IC (50) values of 17.5, 11.4 and 18.8 microM, respectively. Compound 6 exhibited moderate cytotoxicity against HO-8910 cells with an IC (50) value of 17.9 microM.

[structure: see text] [structure: see text] Sesquiterpene lactones, ligulolide A (1) and 6beta,8alpha-dihydroxy-1-oxoeremophila-7(11),9(10)-diene-12,8-olide (2), were isolated from Ligularia virgaurea spp. oligocephala. Their structures were established by analysis of one- and two-dimensional NMR data. The relative stereostructures were determined on the basis of NOESY and single-crystal X-ray experiments. 1 represents a novel sesquiterpene carbon framework, and a possible biosynthetic process is proposed. 2 is a novel eremophilane-type sesquiterpene.

From an extract of the whole plant of Ligularia virgaurea ssp. oligocephala, three new eremophilane sesquiterpene lactones, 10alpha-hydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide (1), 6alpha,10alpha-dihydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide (2), 6beta,10beta-dihydroxyeremophila-7(11),8(9)-dien-12,8-olide (3), as well as four known sesquiterpene lactones with the same carbon skeleton, named toluccanolides A-C (4, 5, 7) and 6beta-hydroxy-8alpha-methoxyeremophila-1(10),7(11)-dien-12,8beta-olide (6) were isolated. Their structures were elucidated by spectroscopic methods including intensive 2D NMR techniques (gCOSY, gHMQC, gHMBC and 1H-1H NOESY for 2) and HR-ESI-MS.

A replication-defective form of chimpanzee adenovirus type 68 (C68) has been developed to circumvent problems posed by widespread preexisting immunity to common human adenovirus vectors. To investigate the determinants of C68 tropism, its interaction with the coxsackievirus and adenovirus receptor (CAR) was studied. Although CHO cells were resistant to transduction by C68 as well as by adenovirus type 5 (Ad5), CHO cells expressing either human or murine CAR were transduced readily. C68 transduction, like Ad5 transduction, was blocked when cells were exposed to anti-CAR antibody or when virus was exposed to a soluble form of the CAR extracellular domain. These results indicate that gene delivery by C68 occurs by a CAR-dependent mechanism.

In this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant.

We previously reported a rare germline variant (c.1-6531) that resulted in allele-specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.

Objective: Sustained antiretroviral therapy (ART)-mediated viral suppression restores responses to vaccination in HIV-1-infected individuals. As ART interruption occur frequently in resource-constrained settings, we studied their effects on the ability to mount humoral immune responses against a neoantigen. Design: Treatment-naive HIV-1-infected individuals were treated with stavudine, lamuvidine and lopinavir/ritonovir. Individuals who maintained viral load less than 50copies/ml and CD4 þ T-cell counts more than 450cells/ml for 6 months received three doses of rabies vaccine, and were randomized to 72 weeks of continuous ART (arm 1) or sequential 2, 4 and 8-week ART interruptions (arm 2). An additional vaccine dose was administered at study end. Methods: Neutralizing antibody titers to rabies virus were assessed in plasma with a rapid fluorescent focus-inhibiting test. Results: The proportion of participants achieving protective (>0.5IU/ml) antibody titer after vaccination was similar (arm 1 ¼92%; arm 2 ¼91%), but over time the cumulative proportion of observations with protective titer was greater in arm 1 than arm 2 (P ¼0.0177). From week 26 after vaccination, antibody titers were lower in arm 2 than arm 1, and volunteers in arm 2 lost protective antibody titers at a greater rate (P ¼0.0029). After boosting, 100% of arm 1 and 95% arm 2 volunteers achieved protective antibody titer. Conclusion: Our data indicate that individuals undergoing recurring ART interruption retain lower neutralizing antibody titers to a neoantigen, but maintain the ability to mount secondary responses upon boosting, suggesting that they might benefit from vaccine schedule intensification. 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2012, 26:1355‐1362

Recombinase-mediated cassette exchange (RMCE) is a powerful method for achieving gene targeting repeatedly at a single mammalian locus. This approach could be applied to the efficient establishment of genetically related cell lines harboring different p53 mutations found in human tumors. To this end we generated a mouse strain called p53 Platform mice (PLF mice), containing PhiC31 integrase-specific attP sequences at the Trp53 locus. The attP sites flank a PGK-neo cassette that has replaced exons 2 to 9 of the endogenous murine p53 gene, generating a null allele. Electroporation of a fluorescence indicator plasmid into embryonic stem (ES) cell lines from PLF mice demonstrated that PhiC31 integrase-mediated cassette exchange (IMCE) can be achieved at60% efficiency without selecting against random insertion. To produce somatic cell lines with endogenously controlled expression of mutant p53, we performed IMCE in PLF murine embryonic fibroblasts (MEFs) with plasmid constructs containing human p53 gene sequences carrying specific tumor-associated missense mutations (A138V; G245S). The MEF cell lines produce the expected mutated mRNA transcripts and express p53 protein that is phosphorylated at serine 15 following DNA damage. Within a few weeks one can thus acquire a family of p53 mutant cell lines from the same population of primary cells, but each harboring a different mutation.

Six new sesquiterpene lactones, scorzoaustriacoside (1), scorzoaustriacin (2), scorzoaustriacin 3-O-beta-d-glucoside (3), 4-epi-dihydroestafiatol (4), 14-isovaleroxyscorzoaustricin (5), and 14-isovaleroxyscorzoaustricin sulfate (6), along with five known guaianolides, were isolated from an acetone extract of the roots of Scorzonera austriaca. The structures of the new compounds were elucidated mainly by interpretation of their 1D and 2D NMR and HRMS data. Several isolates obtained in this investigation were evaluated against a small panel of cancer cell lines.

Indole alkaloids, oxytrofalcatins A-F (1-6), together with five other known alkaloids (7-11), were isolated from the roots of Oxytropis falcata. Their structures were elucidated by comprehensive spectroscopic analyses, including using 1D and 2D NMR spectroscopy and mass spectrometry. This is the first report of N-benzoylindoles from a natural source. Compounds 1-6 lacked significant cytotoxicity against SGC-7901 and HL-60 tumor cell lines.

Cryptococcal meningitis is the most common life-threatening fungal infection and is associated with high mortality in children. Amphotericin B plus flucytosine and fluconazole is the optimal current therapy. Implantation of an Ommaya reservoir for intraventricular infusion of medication and aspiration of cerebrospinal fluid (CSF) for the treatment of increased intracranial pressure (ICP) has been reported. Intraventricular injection of amphotericin B through an Ommaya reservoir in children with cryptococcal meningitis has not been reported previously. We report two children who had cryptococcal meningitis and associated increased intracranial pressure, and were treated with an Ommaya reservoir. Both patients experienced rapid reversal of symptoms. At the time of discharge both patients had recovered and have remained asymptomatic.

AIM: To study mutations in the P-type ATPase (ATP7B) gene responsible for Wilson disease (WD) in the Eastern Chinese population, and the possible correlation of specific mutations with clinical characteristics. METHODS: Mutations of the ATP7B gene were sought by means of direct sequencing in 50 Eastern Chinese WD patients of Han ethnic origin. RESULTS: Two novel mutations, Asp96Gly and Asp196Glu, were first identified. We also compared the characterization of mutations in ATP7B with the clinical findings, and a significant correlation with hepatic manifestations between patients carrying the Arg778Leu mutation and those without was found. CONCLUSION: Gene sequencing analysis was shown to have a high detection rate and accuracy. It may become the first priority in screening of WD patients.

A replication-defective chimeric vector based on the chimpanzee adenovirus serotype C1 was developed and tested as a vaccine carrier in mice. The AdC1 virus is closely related to human adenoviruses of subgroup B2 and uses CD46 for cell attachment. To overcome poor growth of E1-deleted AdC1 vectors on cell lines that provide the E1 of adenovirus of the human serotype 5 (AdHu5) virus in trans, the inverted terminal repeats and some of the early genes of AdC1 were replaced with those from AdC5, a chimpanzee origin adenovirus of subfamily E. The chimeric AdC1/C5 vector efficiently transduces CD46-expressing mouse dendritic cells (DCs) in vitro and initiates their maturation. Transduction of DCs in vivo is inefficient in CD46 transgenic mice. The AdC1/C5 vector induces transgene product-specific B- and CD8(+) T-cell responses in mice. Responses are slightly higher in wild-type mice than in CD46 transgenic mice. Transgene product-specific T-cell responses elicited by the AdC1/C5 vector can be increased by priming or boosting with a heterologous adenovirus vector. Pre-existing immunity to adenovirus of the common human serotype 5 does not affect induction of cell-mediated immune responses by the AdC1/C5 vector. This vector provides an additional tool in a repertoire of adenovirus-based vaccine vectors.

Two new 8,9-seco-cacalol-type sesquiterpene lactones, ligulolide C (1 and 2) and six known sesquiterpenes, adenostylide (3 and 4), cacalol (5), 1 -hydroxy-2-(3'-pentenyl)-3,7-dimethylbenzofuran (6), 1beta, 6alpha-dihydroxy-4(14)-eudesmene (7) and (+)-oplopanone (8), have been isolated from an extract of the whole plant of Ligularia virgaurea spp. oligocephala. The structures of 1 and 2 were confirmed by spectroscopic methods including 1D and 2D NMR spectra. A discussion of biogenesis of 1 and 2 was described. Cytotoxicity against selected cancer cells human promyelocytic leukemia (HL-60), human ovarian (HO-8910) and human lung epithehial (A-549) of compounds 1-8 were measured in vitro.

The mechanism through which cutaneous papillomaviruses induce lesions is largely unknown. Ectopic expression of the DeltaNp63alpha isoform highly increased the viral promoter activity. The co-expression of c-Jun mediated and increased the DeltaNp63alpha activity by binding to the AP-1 site in an enhancer region of the HPV 20 URR. This strong activation by DeltaNp63alpha is diminished in the presence of wtp53 and abolished by the simultaneous expression of "hot-spot" mutant p53 R248W. We demonstrate that c-Jun is responsible for the viral promoter activation through its direct interaction with both DeltaNp63alpha and wtp53. The downregulation by p53 mutant R248W is accompanied by reduced protein levels of DeltaNp63alpha and phosphorylated c-Jun. The data presented in this study provide insight into a possible mechanism through which these cellular proteins may modulate a cutaneous papillomavirus genome to induce viral replication, latent infection or malignant transformation.

Two triple immunization vaccine regimens with adenoviral vectors with E1 deleted expressing Gag of human immunodeficiency virus type 1 were tested for induction of T- and B-cell-mediated-immune responses in mice and in nonhuman primates. The vaccine carriers were derived from distinct serotypes of human and simian adenoviruses that fail to elicit cross-neutralizing antibodies expected to dampen the effect of booster immunizations. Both triple immunization regimens induced unprecedented frequencies of gamma interferon-producing CD8 + T cells to Gag in mice and monkeys that remained remarkably stable over time. In addition, monkeys developed Gag-specific interleukin-2-secreting T cells, presumably belonging to the CD4 + T-cell subset, and antibodies to both Gag and the adenoviral vaccine carriers.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371–393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an α-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Myotonia congenita (MC) is a genetic disease characterized by mutations in the muscle chloride channel gene (CLCN1). To date, approximately 130 different mutations on the CLCN1 gene have been identified. However, most of the studies have focused on Caucasians, and reports on CLCN1 mutations in Chinese population are rare. This study investigated the mutation of CLCN1 in two Chinese families with MC. Direct sequencing of the CLCN1 gene revealed a heterozygous mutation (892G>A, resulting in A298T) in one family and a compound heterozygous mutations (782A>G, resulting in Y261C; 1679T>C, resulting in M560T) in the other family, None of the 100 normal controls had these mutations. Our findings add more to the available information on the CLCN1 mutation spectrum, and provide a valuable reference for studying the mutation types and inheritance pattern of CLCN1 in the Chinese population.

Peptides 31D and VF13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68-76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55-64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, beta-linked sugars in mid-chain positions had a greater inhibitory effect than alpha-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the beta-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of beta-linked carbohydrates might be superior to the addition of alpha-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant alpha-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the alpha- and beta-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.