Adenosine deaminase activity in pleural effusions of lymphoma patients.

BACKGROUND AND AIM: The purpose of this study was to assess the relationship of pleural adenosine deaminase (P-ADA) and non-Hodgkin's lymphoma (NHL). DESIGN AND METHODS: We retrospectively analysed 63 NHL patients with pleural effusions who accepted a diagnostic thoracentesis and who had P-ADA available at the China Medical University Hospital (Taichung, Taiwan) between January 2003 and April 2012. RESULTS: There were 46 exudates [40 malignant pleural effusions (MPE), 5 complicated para-pneumonic effusions and 1 undiagnosed effusion] and 17 transudates. The P-ADA activity was significantly different between the two groups (P < 0.005). Among 40 MPE cases, 29 were due to B-cell and 11 due to T-cell NHL. There was no pleural transudative effusion with P-ADA value higher than 26 U/l in our study, but simultaneously 48% (22/46) of exudative pleural effusions showed a P-ADA value under that cut-off point. The P-ADA level reached the diagnostic cut-off for tuberculosis (40 IU/l) in 11 cases of MPE (11/40 = 27.5%): 9 B-cell NHL (9/29 = 31%) and 2 T-cell NHL (2/11 = 18%). The median levels (25th, 75th percentiles) of P-ADA were 28 IU/l (14-50) in the MPE of B-cell NHL and 26 IU/l (14-28) in the T-cell NHL (P = 0.693). CONCLUSIONS: The use of P-ADA in NHL effusion could aid the separation of transudates from exudates. Around one-quarter MPE of NHL had abnormal P-ADA ( > 40 IU/l). There was no difference in the P-ADA activity in T-cell and B-cell NHL.

Gracilaria bursa-pastoris (Gmelin) Silva extract attenuates ultraviolet B radiation-induced oxidative stress in human keratinocytes.

The purpose of this study was to assess the protective effects of an ethanol extract derived from the red alga Gracilaria bursa-pastoris (Gmelin) Silva (GBE) on ultraviolet B (UVB)-irradiated human HaCaT keratinocytes. GBE exhibited scavenging activity against intracellular reactive oxygen species that were induced by either hydrogen peroxide or UVB radiation. In addition, both the superoxide anion and the hydroxyl radical were scavenged by GBE in cell-free systems. GBE absorbed light in the UVB range (280-320 nm) of the electromagnetic spectrum and lessened the extent of UVB-induced oxidative damage to cellular lipids, proteins, and DNA. Finally, GBE-treated keratinocytes showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies. These results suggest that GBE exerts cytoprotective actions against UVB-stimulated oxidative stress by scavenging ROS and absorbing UVB rays, thereby attenuating injury to cellular constituents and preventing cell death.

High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen.

The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10-30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis.

Vector competence of Culex pipiens molestus (Diptera: Culicidae) from Taiwan for a sympatric strain of Japanese encephalitis virus.

Laboratory strains of Culex pipiens molestus Forskal and Culex tritaeniorhynchus Giles from northern Taiwan were compared for their susceptibility to the Sanhsia MQ1-2 (SH) strain of Japanese encephalitis (JE) virus isolated from Taiwan. After feeding on a sweetened blood-virus mixture, viral titers in Cx. p. molestus during the 14-d incubation period ranged from a minimum of 2.9 log10PFU (plaque forming units) per mosquito on day 3 after ingestion to a maximum of 4.65 log10PFU at day 8 and in Cx. tritaeniorhynchus from 2.6 on day 10-5.18 log10PFU per mosquito on day 13. Although virus titer in Cx. p. molestus was lower than in Cx. tritaeniorhynchus at the end of the experiment, this difference was not statistically significant. The median infective dose (ID50) for Cx. p. molestus was 2.83 log10PFU and for Cx. tritaeniorhynchus was 1.02 log10PFU per mosquito, and this difference also was not significant. There also was no significant difference between the median infective dose for transmission (TID50) per mosquito for Cx. p. molestus (5.34 log10PFU) and Cx. tritaeniorhynchus (4.59 log10PFU). We concluded that Cx. p. molestus is an effective laboratory vector of JE virus.

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae).

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.

Identification of free deaminated sialic acid (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in human red blood cells and its elevated expression in fetal cord red blood cells and ovarian cancer cells.

Chemical studies have shown the occurrence of the deaminated sialic acid 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) in paired samples of blood obtained from mothers and newborns of healthy human individuals. Most of the KDN was found in red blood cells, although low levels were detected in mononuclear cells. No N-glycolylneuraminic acid was detected. Unexpectedly, nearly all of the KDN in fetal cord and matched maternal red blood cells was present as the free sugar and comparatively little occurred conjugated or as cytidine 5'-KDN phosphate. The amount of free KDN in fetal newborn red blood cells was 2.4-fold higher than in red blood cells from the mothers or from healthy nonpregnant women. Free KDN was also identified in normal human ovaries, in ovarian tumors, and in ascites cells obtained from ovarian cancer patients. Importantly, as in fetal cord red blood cells, a distinguishing feature of KDN expression in ovarian tumor cells was an elevated level of free KDN compared with normal controls. A positive correlation was found between an increase in the ratio of free KDN/N-acetylneuraminic acid in ovarian adenocarcinomas and the stage of malignancy. This was particularly evident in tumor cells isolated from the ascites fluid. The central importance of these new findings is 2-fold. First, they show that free KDN is a minor but ubiquitous sialic acid in human red blood cells and that its elevated expression in red blood cells from fetal cord blood compared with maternal red blood cells may be developmentally related to blood cell formation during embryogenesis. Second, the enhanced expression of KDN in ovarian cancer cells suggests that this sialic acid, like the alpha2,8-linked polysialic acid glycotope, may be an oncofetal antigen in these tumors and thus could be an "early warning" signal for onset of disease and/or a marker for detection of recurrence of disease. These new findings highlight the importance of elucidating the role that KDN and KDN-containing glycoconjugates may play in normal development and malignancy.

Round-spotted pufferfish (Tetraodon fluviatilis) snf5 gene is oriented in a tail-to-tail manner with the set gene which encodes an inhibitor of protein phosphatase 2A.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish Fugu rubripes rubripes (Fugu). Due to its compact genome and small introns, Fugu has been introduced as a model for genome studies. Recently, the round-spotted pufferfish has also been proposed as a new model for genome studies because of the ease in obtaining material and high-sequence homology to that of Fugu. In this study, we have cloned and characterized the snf5 and set genes from the round-spotted pufferfish. The snf5 gene is composed of 9 exons spanning about 2.9 kb whereas the set gene consists of 8 exons spanning about 2.7 kb. They are linked in a tail-to-tail manner with an intergenic region of about 6.5 kb. So far, the genomic structures of human snf5 and set genes are unknown. Based on our data, the pufferfish SNF5 and SET display high amino acid sequence identity (>90%) with the respective human genes. By primer extension and sequence analysis, we found that putative promoter region of the snf5 gene contains a typical TATA box and numerous potential binding sites for transcription factors including AP1, AP2, AP3, c-Myb, HNF-5, and NF-IL6. As for the set gene, its promoter region does not have any TATA or CCAAT motif and contains a few potential binding sites for transcriptional factors such as c-Myb and gamma-IRE. When these promoter regions were placed upstream of the CAT reporter gene and transfected into a carp CF cell line, the 5'-upstream 1.6-kb DNA fragment of the snf5 gene displayed stronger promoter activity, approximately three-fold higher than that of the 5'-upstream 1.3 kb DNA fragment of the set gene. By transient expression and immunofluorescent staining, we also showed that the pufferfish SNF5 and SET are nuclear proteins, consistent with their postulated roles as transcriptional factors.

Genomic organization and characterization of the promoter region of the round-spotted pufferfish (Tetraodon fluviatilis) JAK1 kinase gene.

Seventeen kilobases of genomic DNA containing the promoter and the coding region of the round-spotted pufferfish JAK1 gene was isolated and completely sequenced. This gene consists of 25 exons and 24 introns spanning about 13.5 kb, compared to > 30kb in carp JAK1 gene. Primer extension analysis revealed one transcription initiation site which was 376 bp upstream of the translation initiation site. The sequence of the 2.9 kb region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including HNF-5, GCF, Sp1, CRE, AP2, GATA, GAGA, E2A, p53, and NF-IL6. When this region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme three times more efficiently than could the common carp JAK1 promoter.

In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3.

We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.