Pediatric Quality of Life Inventory (PedsQL) 3.2 Diabetes Module for youth with Type 2 diabetes: reliability and validity.

AIM: To test the measurement properties of the revised and updated Pediatric Quality of Life Inventory (PedsQL) 3.2 Diabetes Module originally developed in Type 1 diabetes in youth with Type 2 diabetes. METHODS: The PedsQL 3.2 Diabetes Module and PedsQL Generic Core Scales were administered in a field test study to 100 young people aged 9-25 years with Type 2 diabetes. Factor analysis was conducted to determine the factor structure of the items. RESULTS: The 15-item Diabetes Symptoms Summary Score and 12-item Type 2-specific Diabetes Management Summary Score were empirically derived through factor analysis. The Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores showed acceptable to excellent reliability across the age groups tested (α = 0.85-0.94). The Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores evidenced construct validity through large effect size correlations with the Generic Core Scales Total Scale Score (r = 0.67 and 0.57, respectively). HbA1c was correlated with the Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores (r = -0.13 and -0.22). Minimal clinically important difference (MCID) scores were 5.91 and 7.39 for the Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores. CONCLUSIONS: The PedsQL 3.2 Diabetes Module Diabetes Symptoms Summary Score and Type 2-specific Diabetes Management Summary Score exhibited satisfactory measurement properties for use as youth self-reported diabetes symptoms and diabetes management outcomes for clinical research and clinical practice for young people with Type 2 diabetes.

Monitoring Taiwanese bovine arboviruses and non-arboviruses using a vector-based approach.

In current sampling approaches, there exists a divergence between the surveillance of arthropod-borne and that of non-arthropod-borne viruses. It is commonly held that the collection of vector specimens applies only to arbovirus surveillance and that the surveillance of non-arboviruses must rely on traditional methods that involve the sampling of blood, faeces or saliva, or other examinations. The vector-based approach is a sampling method that has the ability to survey both arboviruses and non-arboviruses by distinguishing engorged vector specimens from entire vector samples. Accordingly, five arboviruses and three non-arboviruses were detected in a study using a vector-based approach conducted during 2012-2015. Hence, this report provides the first description of the Taiwanese vector species for the bovine arboviruses detected. The present investigations demonstrate that the vector-based approach applies not only to the surveillance of arboviruses, but also has potential as a possible tool for monitoring non-arboviruses on livestock farms in the future.

Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification.

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

Expression in Escherichia coli of open reading frame gene segments of HTLV-III.

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.

Vitamin K3-induced cell cycle arrest and apoptotic cell death are accompanied by altered expression of c-fos and c-myc in nasopharyngeal carcinoma cells.

Vitamin K3 is known to inhibit the growth of various rodent and human tumor cells. However, the molecular mechanism of its action is still elusive. We have found that vitamin K3 induces cell cycle arrest and apoptotic cell death in nasopharyngeal carcinoma (NPC) cells, as evaluated by flow cytometry and DNA gel electrophoresis. Involvement of c-fos and c-myc proto-oncogenes and expression of their proto-oncoproteins in VK3-induced cell cycle arrest and apoptosis were also investigated. Northern blot analysis of NPC cells treated with 50 microM VK3 showed that c-fos was transiently induced for 60 min after treatment, while c-myc was persistently induced for 1-9 h after drug treatment. Western blot analysis also showed that c-Fos was induced at 4-6 h and at 1-4 h after treatment with 50 microM and 200 microM VK3 respectively, while c-Myc was induced at 1-6 h and at 4-6 h, respectively, after such treatments. These results suggest that the expression of c-fos and c-myc may play an important role in the signaling mechanism of VK3-induced growth arrest and apoptotic cell death.

Detection and purification of a recombinant human B lymphotropic virus (HHV-6) in the baculovirus expression system by limiting dilution and DNA dot-blot hybridization.

A recombinant virus Ac373-HB was detected and purified from the transfection mixture of wild type and recombinant virus in the baculovirus expression system using a combination of limiting dilution and DNA dot-blot hybridization. This method allows for a quick and convenient way of detection and purification of recombinant virus without the need to use a plaque purification step. It is generally applicable to other expression systems besides the baculovirus system described here.

Evaluation of cyano bonded phases for the fractionation of bioactive complex mixtures.

Cyanopropyl bonded-phase sorbents are investigated for the liquid chromatographic (LC) fractionation of complex mixtures with the goal of extending routine sample fractionation to samples containing highly polar biologically active components. Separations based both on gravity-flow column chromatography and on high performance liquid chromatography (HPLC) are evaluated for efficiency and resolving power. Typical gravity-flow column separations of neutral to moderately polar mixtures are found to be less effective than those employing traditional sorbents; however, HPLC methods could be called upon to provide the resolving power necessary for satisfactory separations of these mixtures. For mixtures of polar reference compounds, cyano bonded phases provide good separations and are very efficient, permitting the elution of components too polar to be recovered from traditional sorbents. A scheme combining gravity-flow chromatography with HPLC is developed for the fractionation of complex mixtures into compound classes. It is based on the chromatographic behavior of reference compounds covering a wide polarity range utilizing cyanopropyl sorbents exclusively. Results are presented for the fractionation of two air particulate reference samples containing highly polar components.

Integrated control of the dengue vector Aedes aegypti in Liu-Chiu village, Ping-Tung County, Taiwan.

Because of an inadequate supply of potable water, villagers of Small Liu-Chiu Isle, Ping-Tung County, Taiwan, store water in containers supporting a large population of Aedes aegypti. In 1989-96, integrated control measures against Ae. aegypti were implemented on the basis of community participation. These measures included release of mosquito larvivorous fish in the drinking water storage facilities, application of larvicides to the water storage facilities in vegetable gardens, removal of discarded and unused containers and tires, improvement of household water storage facilities, and increase of potable water supply. Before implementation of the integrated control measures in 1988, 74% of the water-containing vessels were water storage facilities, and 24% of those were infested by Ae. aegypti. In 1989, the Breteau index for the entire island, indicating the average distribution density for larval Ae. aegypti, was 53.9, as compared to an index of 1.2 in 1996. In 4 villages located at the southwest and middle of the island, Ae. aegypti nearly became extinct because of the enthusiastic participation of the community. Before the implementation of integrated control, Ae. aegypti was the dominant species in containers both inside and outside the household, but after the integrated control, Aedes albopictus became predominant outside.

Framework for application of geographic information system to the monitoring of dengue vectors.

In a successful management program of dengue vectors, not only health education, source reduction or insecticide application should be conducted, but all basic information should also be manipulated properly and efficiently. This information includes the surveys of species, dispersal and dynamics of vectors, as well as the detection of breeding sources, and the records of dengue cases and epidemic periods. Most of the above information expressed as regionalized variables always varies spatially and/or temporally. However, due to the deficiency of topological information, the conventional database management system cannot efficiently analyze those dengue related data. Thus, we have applied the geographic information system (GIS) to the monitoring of dengue vectors. The purpose of this report is to introduce the basic concepts of GIS, to describe the framework of the prototype dengue vector monitoring system which was built using data collected from the Sanmin area, Kaoshiung city, Taiwan, and to indicate the possibility of using this system to manipulate spatially correlated data and support decision making in the control of dengue disease.

The heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases: evidence from the effects of okadaic acid.

We examined the effects of okadaic acid (OA), a potent and specific inhibitor of serine phosphatases 2A and 1, on the transient expression of an hsp 70 promoter-reporter gene construct in IMR-90 human diploid lung fibroblasts. We showed that OA markedly potentiated the heat-induced but not the basal expression of pHBCAT, a full-length human hsp-70-promoter-driven CAT gene construct. This effect of OA was dose and time dependent and promoter specific. Importantly, the potentiating effects of OA appeared to be independent of the binding of the activated heat shock transcription factor (HSTF) to its consensus DNA sequence, the heat shock element (HSE). Thus, OA had no effect on the HSTF DNA-binding activity as measured by mobility shift assay, and mutation of the HSE sequence did not obliterate the stimulatory effects of OA on reporter gene expression under a heat shock condition, although heat shock by itself was without effect. Analysis of the status of phosphorylation of the largest subunit of RNA polymerase II provided evidence that this effect of OA is attributable, at least in part, to the increased phosphorylation of RNA polymerase II. These results provided evidence that the heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases. We propose that the heat-induced transcriptional activation of hsps is associated with phosphorylation of component(s) of the transcription complex; one of the likely candidates being the transcriptionally engaged RNA polymerase II. OA, by inhibiting phosphatase 2A and 1 activity, enhanced this phosphorylation and potentiated the transcriptional activation of hsps.

Okadaic acid markedly potentiates the heat-induced hsp 70 promoter activity.

The effects of okadaic acid (OA), a potent and specific inhibitor of serine/threonine phosphatases 2A and 1, on the transient expression of a human hsp 70 promoter-linked chloramphenicol acetyltransferase gene transfected into N-18 mouse neuroblastoma cells were determined. Assays of reporter gene activity showed that nanomolar concentrations of OA markedly potentiated the heat-induced (but not the basal) expression of pHBCAT, a full-length hsp 70 promoter-driven chloramphenicol acetyltransferase gene construct. This effect of OA was dose-dependent and promoter-specific and appeared to be attributable to inhibition of protein phosphatase 2A as opposed to protein phosphatase 1. The ability of OA to potentiate the heat-induced expression of pHBCAT appeared to be a feature common to several different cell types examined. We propose that the heat-induced transcriptional activation of heat shock genes is associated with the phosphorylation of component(s) of the transcription complex and that OA enhances this phosphorylation, thereby potentiating the heat-induced hsp 70 promoter activity.

Synthesis of a human leukocyte interferon with a modified carboxy terminus in Escherichia coli.

A recombinant plasmid was constructed that results in deletion of eleven COOH-terminal amino acids of human leukocyte A interferon and their replacement by nine unrelated amino acids encoded by the beta-lactamase gene of pBR322. This altered human leukocyte interferon exhibits antiviral activity slightly lower than the natural molecule and appears to be more susceptible to proteolytic degradation as well. These results also confirm the conclusion that the eleven COOH-terminal amino acids are not essential for antiviral or antiproliferative activity.

Identification and characterization of human immunodeficiency virus type 1 gag-pol fusion protein in transfected mammalian cells.

Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.

Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells.

HTLV-III, the etiological agent of the acquired immunodeficiency syndrome, contains in its genome coding regions for several novel proteins. One of these, the 3' open reading frame (3'orf) encodes proteins of 26-27 kDa which are expressed in infected cells both in vivo and in vitro. A specific antiserum has been raised against the recombinant 3'orf protein synthesized in bacteria and used to localize the viral proteins by subcellular fractionation and immunofluorescence on HTLV-III infected cells. The antiserum specifically immunoprecipitated the 26- to 27-kDa proteins from both the cytoplasmic (S100) and the membrane fractions, with an enrichment in the latter. The proteins were not detected in the nucleus or organelle (S100 pellet) fractions. These proteins were also recognized in the same subcellular fractions by human sera from patients with AIDS. Indirect immunofluorescence on fixed infected cells confirmed the presence of the proteins in the cytoplasm. Immunoprecipitation and Western blot analysis of total proteins from disrupted HTLV-III virions with the specific antiserum failed to detect the 3'orf protein products, suggesting that they are not a major component of mature virions and may be involved in the intracellular regulation of viral replication.

Identification, cloning, and characterization of an immune activation gene.

We have identified an immune activation gene, denoted Act-2, by differential hybridization screening of an activated T-cell library. The gene is induced rapidly after T-cell activation with phytohemagglutinin, B-cell activation with Staphylococcus aureus Cowan I, and monocyte activation with lipopolysaccharide. We have isolated a cDNA containing the full-length coding region. The deduced amino acid sequence predicts an open reading frame of 92 amino acids, including a very hydrophobic N terminus, which by weight matrix score is predicted to be a signal peptide. Using a baculovirus expression system, we have shown that this gene encodes a secreted product. It is therefore possible that Act-2 represents a newly discovered cytokine.

A cDNA clone encoding a product of activated human T lymphocytes.

A cDNA cloning approach was used to study the regulation of gene expression in human T lymphocytes upon mitogen stimulation. Poly(A)+ mRNA was prepared from phytohemagglutinin A (PHA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) activated human T cells and a cDNA clone library was constructed. After screening by colony hybridization with [32P]cDNA probes made from resting and activated T cell mRNA, several clones whose mRNA increased at least 10 to 20-fold upon stimulation were isolated. Northern blot analysis of the mRNA from various cell types using these cDNA clones as probes revealed that one of the cDNA clones, pNC5A, encoded a gene expressed only in PHA and TPA-stimulated human T lymphocytes and in a human neoplastic T cell line HUT102-SH9. Less than 20 copies of this mRNA species per cell was detected in resting human T lymphocytes, B lymphocytes and monocytes and in two other T cell lymphoma lines (CEM and MOLT4), two B lymphoblastoid cell lines (WIL2-729-HF2 and HFB-1), a myeloid cell line (HL60) and a human embryonic lung fibroblast cell line (MRC-5). Hybrid selection translation and sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis of the translated product indicated that a polypeptide of 30,000 to 32,000 Mr is encoded by this particular cDNA clone. Thus, this cDNA clone may define a novel gene that is expressed only in activated human T cells.

Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity.

It is generally believed that the gag gene product of human immunodeficiency virus type 1 (HIV-1) is processed into several core proteins by a virus-specific protease. We used deletion mutation analysis to study the role of HIV-specific protease in the processing of core proteins and its requirement for viral infectivity. Several mutant genomes with deletions in the protease gene were constructed. A mammalian cell line, COS-M6, transfected with the wild-type viral genome was shown to produce virions containing processed core proteins, while COS-M6 cells transfected with two mutated genomes could express only the core protein precursor, Pr56gag. The wild-type transfectant produced infectious virus; both transfectants expressing the mutated genomes also produced virions, and one of them still retained reverse transcriptase activity. However, the mutant viral particles were devoid of infectivity. Virions with a distinct central core and an electron-dense nucleoid budded out from the plasma membrane of COS-M6 cells transfected with the wild-type genome. In contrast, noninfectious virions that budded either into cytoplasmic vacuoles or out from the plasma membrane of COS-M6 cells transfected with mutant genomes contained ring-shaped nucleoids. These results indicate that the HIV-1 protease plays a role not only in the maturation of the core proteins but also in the assembly of the virus and thus is required for viral infectivity.

Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera.

The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames. To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector. The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins. Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo. A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein. The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes.

Tissue-specific cancer-related serpin gene cluster at human chromosome band 3q26.

Approximately one quarter of the identified human serpin genes are cancer-related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas-specific protease inhibitor, pancpin (PI14), and the brain-associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov-serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor-1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33-35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down-regulated in tumor brain tissues and in two brain cancer cell lines: U-87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI-10077 (telomere) is located downstream of the 3'-flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is approximately 100 kb, and hence we speculate that other tissue-specific cancer-related serpin genes are likely to reside within this 3q26.1 cluster region.